Bu makalenin yayın Tennessee Üniversitesi Araştırma Dairesi bir hibe ile desteklenmiştir. Yazarlar, öğrenciler ve araştırmacılar, bu işlemler rafine için çalıştılar, önceki nesil teşekkür ederim. Araştırma, Ulusal Bilim Vakfı (NSF-0.851.113, NSF 0.825.405 ve NSF 0.550.485) hibe tarafından desteklenmiştir.

1. &quot;Virüs-free&quot; Su (Wilhelm &amp; Poorvin 2001) oluşturun için Deniz Suyu Ultrafiltrasyon <ol><li> Deniz suyu / lakewater yaklaşık 20L mümkün olduğunca aseptik toplanır. </li><li> Su, 142 mm çaplı polikarbonat seri ile toplum analizi için -20 ° C&#39;de muhafaza edilebilir 0,8 mikron filtre prefiltered. Daha büyük gözenek boyutu filtreleri, çok verimli sistemler için bu adımı önce kullanılıyor olabilir. </li><li> 30 kDa kesme spiral kartuşu tüm virüsleri dışlamak için kullanılan bir Amicon M12 sistemi (Millipore), hatta küçük RNA virüsleri Ultra filtre su elde etmek için. </li><li> Yaklaşık 15-16 kPa backpressure örnekleri ile işlenmiş ve ~% 25 hızda yoğunlaşmıştır. </li><li> ~ 500 ml su kalan örnek virüs serbest su, geri kalan 19.5 L viral üretim deneyleri için kullanılan olacak konsantre bir virüs topluluk (diğer deneyler için kaydedilebilir) içerecektir. </li><li> Amicon M12 sistemi kullanımı her gün sonra, membran filtre kartuşunun zarar görmesini önlemek için temizlenmesi gerekir. </li><li> Deniz suyu ile çalışıyorsanız, 30-45 dakika için 0,1 N NaOH çözeltisi ile yıkanması takip Milli-Q su en az 6L membran yıkayın. </li><li> Yine kartuşu en az 6L Milli-Q su ile durulayın. </li><li> M12 sistemini kullanarak bittiğinde, spiral kartuşu, 4 0.05MH 3 PO 4 çözüm ° C saklanmalıdır </li></ol> 2. Viral Üretim Yöntemi Virüs Azaltma (Wilhelm ve ark 2002) <ol><li> Hem de ev sahibi ve virüsler, 0.2 mikron nominal gözenek boyutu düşük protein bağlayıcı filtresi (örneğin, Durapore) üzerine yerleştirilir. Ile elde edilen ve bir sterifilter ünitede yerleştirilir deniz suyu / lakewater örnek 500 ml </li><li> Sürekli olarak, filtre üzerinde yoğunlaşarak bakteri hücrelerinin inhibe etmek için, steril bir transfer pipet kullanarak örnek resuspending örnek hafifçe &lt;200 mmHg basınçlı vakum. </li><li> Yavaş yavaş ultrafiltratında üç ciltlik önemli ölçüde örnek ücretsiz virüs sayısını azaltmak için bakteriyel süspansiyona eklenir. </li><li> Bakteriyel fraksiyonu virüs ücretsiz su ile 500 mL geri seyreltilir ve her biri 150 ml olmak üzere üçe ayrılır çoğaltır ve açık 250ml polikarbonat şişe konur. </li></ol> 3. Viral Üretimi için Teğetsel Akış Filtrasyon (TFF) Yöntemi (Weinbauer ve ark 2002, Winget ve ark 2005) TFF viral azaltma yöntemine alternatif bir yaklaşımı temsil ediyor. <ol><li> Yukarıda açıklandığı gibi, yaklaşık 500 ml doğal örnek toplanır. </li><li> Bu örnek, gözenek boyutu 0.2 mikron nominal teğetsel akış filtrasyon sistemi kullanarak yoğunlaşmıştır. </li><li> Yaklaşık 10-15 ml bakteri fraksiyon azalır, Ultra filtre, virüs serbest su eklenir ve yukarıdaki gibi dağıtılır. </li><li> Çoğaltmak şişe çevre odaları kullanılarak yerinde koşullarda inkübe edilir. <ol><li> Işık seviyeleri net tarama ile mavi renkli akrilik veya şeffaf akrilik kullanarak ışık yoğunluğunu azaltmak için yüzey koşulları değişmiş. </li><li> Ortam yüzey sıcaklıkları genellikle akan deniz suyu güverte inkübatör ile elde edilir. </li></ol></li><li> Cryovials ilave% 2,0-2,5 steril glutaraldehid son bir konsantrasyon ile 0 zamanında bakteriyel ve viral bolluk tahminleri için numuneler alınır. Bu örnekler hemen sıvı nitrojen ile dondurulur flaş ve işlenmiş kadar dondurularak saklanmalıdır. <ol><li> Sıvı azot mevcut değilse, mikroskopi slaytlar (aşağıdaki yordama bakın) hazırlanan ve hemen işleme olabilir </li></ol></li><li> Alınan örnekler, yukarıda anlatılan yöntemi ile en az 10 saat boyunca her 2.5 saatte toplanır. <ol><li> Bu zaman su kantitatif PCR analizi için toplanan olabilir. 5 ml numune sıvı nitrojen içinde acil flaş dondurma hiçbir sabitleştirici ajan cryovial eklenebilir. </li></ol></li></ol> 4. Viral Üretim Mikroskobu (Noble ve Fuhrman 1998 Wen ve ark 2004) <ol><li> 0.02-mikron mikroskopi için filtre uygulanmasını Donmuş numuneler buz üzerinde çözdürülmelidir. </li><li> SYBR Green, steril su ile stok solüsyonu 01:10 seyreltilmesi ile bir stok solüsyonu hazırlayın. Sonra, stok solüsyonu, stok solüsyonu 1 ml, 39 ml steril su ekleyerek çalışan bir solüsyon hazırlanır. % 50 gliserol,% 50 fosfat serum fizyolojik tamponlu (PBS, 0.05 M Na 2 HPO 4,% 0.85 NaCl, pH 7.5) ve% 2.5 p-fenilendiamin taze stok solüsyonu başlamadan önce hazırlanmalıdır. 4% 50% glycerol/50 PBS çözüm tutun ° C ve P-phenylenediamine stok -20 ° C başlangıç ​​kadar karanlıkta. Sağ filtreleme önce Antifade çözüm olarak kullanılmak üzere bir final konsantrasyon% 0.1% 50% glycerol/50 PBS p-fenilendiamin ekleyin. </li><li> 25 mm 0.45 mikron MicronStep, selülozik destek filtre üst 0.02 mikron Anodisc filtre koyun. Tamamen kurutulmuş kadar 20 pKa Anodisc ve vakum üst sabit numune 850 mcL ekleyin. 100 steril bir Petri kabı SYBR Green çalışma çözüm mcL ve hala vakum, SYBR Green filtre kulesi ve yerden dikkatlice Anodisc kaldırmak. Örnekleri 20 dakika boyunca oda sıcaklığında karanlıkta inkübe edin. Dikkatle SYBR Green çözüm filtreyi kaldırmak ve tüm kalan boya kaldırmak için bir filtre ile Kimwipe arka fitil. İsterseniz, kule filtre dönmek ve aşırı leke durulayın filtre ile 0.02 mikron süzülmüş su veya steril ortam 800 mcL kadar geçmek. </li><li> Bir mikroskop lamı antifade çözüm küçük bir damla ekleyin ve üstüne bir kapak kayma yerleştirin. Kapak kayma çıkarın ve mikroskop lamı antifade çözüm ıslak kuru filtre ekleyin. Yine kapak kayma antifade çözüm küçük bir miktar ekleyin ve yavaş yavaş bu formun herhangi bir kabarcıkları kurtulmak için emin olun, filtrenin üstüne yerleştirin. </li><li> Gereken (bu solmaya önlemek için bir kaç ay içinde kullanılır ve virüs sayıları indirdi olmalıdır) kadar -20 ° C&#39;de hemen slaytlar dondurmak </li><li> Virüsler, geniş bir mavi filtre seti (λ Ex = 450 - 490 nm, λ Em = λ bir bastırma filtresi 510 nm = 510 nm) ile (bizim durumumuzda bir Leica DMRXA mikroskop) floresan mikroskopi kullanılarak numaralandırılır. Her filtre, filtre zarından virüs bile dağılımını sağlamak için her alanda ızgara toplam virüsleri ölçmek için emin olun, en az 20 alan sayılır görünümü olacak. </li><li> Üç bağımsız virüs tekrarını ortalama oranları hesaplanır ve standart sapma üretim oranları belirlenir çoğaltır. </li></ol> 5. Temsilcisi Sonuçlar Araştırmacı tarafından toplanan ham veri virüs bolluk tekrarını oranları üretmek için minimal matematiksel işleme gerektirir. Bu çalışmada kaynaklanan birincil veri seti inkubasyon alınan örnekler virüs bolluk tekrarını oranları. Bu sonuçlar, örneklerin her biri için virüs bolluğu vs zaman bağımsız regresyonlar oluşturur. Her bir numune için ayrı ayrı inkubasyon araştırmacı oranlarının yanı sıra bir varyasyon tahmini (örneğin, standart sapma) (bkz. Şekil 1). Hesaplayabilirsiniz üç çoğaltmak-inkubasyon tamamlayarak, bu yüzden bir tedavi olarak hareket Bu sürecin bir ihtar virüs bolluk değişmez bir azalma örnek virüs yükünü taşıyan ana hücrelerinde azalmaya yol açar. Kaynak (filtrelenmemiş deniz suyu veya göl suyu) ve T = 0 inkübasyon örnek, bakteri bolluk sayımı bu kayıp telafi etmek gereklidir. Bu bilgiler, kayıp hücrelerinin yüzdesini hesap kullanılır: virüs bolluk azaltma süreci için ya da herhangi bir mikrobiyal topluluğun üyelerine karşı seçici olduğunu varsayarak, bu faktör daha sonra virüs yerinde üretim hızı tahmin etmek için kullanılır. . <img src="/files/ftp_upload/2196/2196fig1.jpg" alt="Şekil 1" /> Şekil 1. Epifloresans mikroskopi kullanılarak in situ koşullarda 10 saat inkübasyon üzerinde parçacıklar gibi virüs Örnekleri üretimi 2008 yılı Eylül ayında Yeni Zelanda&#39;nın kıyılarında çiçek fitoplankton sırasında toplanmıştır. <img src="/files/ftp_upload/2196/2196fig2.jpg" alt="Şekil 2" /> Şekil 2. Assaying virüs üretim için iş akış sürecinin şematik diyagramı. Virüssüz su üretmek için numune su ultrafiltrasyon süreci başlar. Bu bir ultrafiltrasyon sistemi kullanılarak tamamlandı. Paralel su örneklerinde mikrobiyal topluluğun (enfekte ve enfekte olmayan hücrelere bir karışımını içeren) muhafaza ederken, bir filtre ile geçti aynı sitede ve serbest virüslerle toplanır. Bu topluluk daha sonra virüs ücretsiz su ile yeniden süspanse ve yerinde koşulları altında inkübe edilir . Virüs tekrarını oranı, önümüzdeki 10 saat sonra virüs üretim oranlarını belirlemek için izlenmektedir.

<ol>
	<li>Higgins, J. L., Kudo, I., Nishioka, J., Tsuda, A., Wilhelm, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+response+of+the+virus+community+to+a+mesoscale+iron+fertilization+in+the+sub-Arctic+Pacific+Ocean.">The response of the virus community to a mesoscale iron fertilization in the sub-Arctic Pacific Ocean.</a> Deep-Sea Res II. 56, 2788-2795 (2009).</li><li>Mioni, C. E., Poorvin, L., Wilhelm, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus+and+siderophore-mediated+transfer+of+available+Fe+between+heterotrophic+bacteria:+characterization+using+a+Fe-specific+bioreporter.">Virus and siderophore-mediated transfer of available Fe between heterotrophic bacteria: characterization using a Fe-specific bioreporter.</a> Aquat Microb Ecol. 41, 233-245 (2005).</li><li>Noble, R. T., Fuhrman, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+SYBR+Green+I+for+rapid+epifluorescence+counts+of+marine+viruses+and+bacteria.">Use of SYBR Green I for rapid epifluorescence counts of marine viruses and bacteria.</a> Aquat Microb Ecol. 14, 113-118 (1998).</li><li>Parada, V., Herndl, G., Weinbauer, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+burst+size+of+heterotrophic+prokaryotes+in+aquatic+systems.">Viral burst size of heterotrophic prokaryotes in aquatic systems.</a> J Mar Biol Assoc U K. 86, 613-621 (2006).</li><li>Poorvin, L., Rinta-Kanto, J. M., Hutchins, D. A., Wilhelm, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+release+of+Fe+and+its+bioavailability+to+marine+plankton.">Viral release of Fe and its bioavailability to marine plankton.</a> Limnol Oceanogr. 49, 1734-1741 .</li><li>Rowe, J. M., Saxton, M. A., Cottrell, M. T., DeBruyn, J. M., Berg, G. M., Kirchman, D. L., Hutchins, D. A., Wilhelm, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constraints+on+virus+production+in+the+Sargasso+Sea+and+North+Atlantic.">Constraints on virus production in the Sargasso Sea and North Atlantic.</a> Aquat Microb Ecol. 52, 233-244 (2008).</li><li>Weinbauer, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecology+of+prokaryotic+viruses.">Ecology of prokaryotic viruses.</a> FEMS Microbiol Rev. 28, 127-181 (2004).</li><li>Weinbauer, M. G., Peduzzi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequency,+size+and+distribution+of+bacteriophages+in+different+marine+morphotypes.">Frequency, size and distribution of bacteriophages in different marine morphotypes.</a> Mar Ecol Prog Ser. 108, 11-20 (1994).</li><li>Weinbauer, M. G., Winter, C., H&amp;ouml, f. l. e., G, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconsidering+transmission+electron+microscopy+based+estimates+of+viral+infection+of+bacterioplankton+using+conversion+factors+derived+from+natural+communities.">Reconsidering transmission electron microscopy based estimates of viral infection of bacterioplankton using conversion factors derived from natural communities.</a> Aquat Microb Ecol. 27, 103-110 (2002).</li><li>Wen, K., Ortmann, A. C., Suttle, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+estimation+of+viral+abundance+by+epifluorescence+microscopy.">Accurate estimation of viral abundance by epifluorescence microscopy.</a> Appl Environ Microbiol. 70, 3862-3867 (2004).</li><li>Wilhelm, S. W., Brigden, S. M., Suttle, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dilution+technique+for+the+direct+measurement+of+viral+production:+a+comparison+in+stratified+and+tidally+mixed+coastal+waters.">A dilution technique for the direct measurement of viral production: a comparison in stratified and tidally mixed coastal waters.</a> Microb Ecol. 43, 168-173 (2002).</li><li>Wilhelm, S. W., Matteson, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Freshwater+and+marine+virioplankton:+a+brief+overview+of+commonalities+and+differences.">Freshwater and marine virioplankton: a brief overview of commonalities and differences.</a> Freshw Biol. 53, 1076-1089 (2008).</li><li>Wilhelm, S. W., Poorvin, L., Paul, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Quantification of algal viruses in marine samples. , 53-66 (2001).</li><li>Wilhelm, S. W., Suttle, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viruses+and+nutrient+cycles+in+the+sea.">Viruses and nutrient cycles in the sea.</a> Bioscience. 49, 781-788 (1999).</li><li>Wilhelm, S. W., Weinbauer, M. G., Suttle, C. A., Jeffrey, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+sunlight+in+the+removal+and+repair+of+viruses+in+the+sea.">The role of sunlight in the removal and repair of viruses in the sea.</a> Limnol Oceanogr. 43, 586-592 (1998).</li><li>Winget, D. M., Williamson, K. E., Helton, R. R., Wommack, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tangential+flow+diafiltration:+an+improved+technique+for+estimation+of+virioplankton+production.">Tangential flow diafiltration: an improved technique for estimation of virioplankton production.</a> Aquat Microb Ecol. 41, 221-232 (2005).</li></ol>

Virüsler deniz mikrobiyal topluluklar nasıl etkilediğini anlamada önemli bir bileşeni virüs parçacık üretilmektedir hızını belirlemek için. Bollukları çoğu sistemlerde statik (Wilhelm &amp; Suttle 1999, Weinbauer 2004) (daha fazla veya daha az) ve sucul sistemlerde bu virüsler hızla kaldırıldı veya non-enfeksiyöz işlenen göz önüne alındığında (Wilhelm ve ark 1998), daha sonra üretim oranları olmalıdır görece hızlı bir kayıp parçacıkları yerine. Mikrobiyal topluluğun neden ölüm virüsleri tahmin, bir virüs, bir hücreye (&quot;patlama boyutu&quot;) lyses her zaman birçok virüs nasıl üretildiği bir bilgi gerektirir. Doğal örneklerinde Virüs patlama boyutları büyük ölçüde değişebilir. Burst boyutları doğrudan transmisyon elektron mikroskobu (örneğin, Weinbauer &amp; Peduzzi 1994) tarafından belirlenen, ancak bu her zaman pratik değil, belirli bir laboratuvar kapasitesinin ötesindeki sık sık ya da olabilir. Ampirik olarak tespit edilemediği durumlarda, parçalayıcı olay başına 24 virüs edebiyat değerleri, tatlı su sistemleri için deniz sistemleri ve 34 için kullanılan (Parada ve ark 2006 ). Bu sayıya bölündüğü virüs üretim oranı ise, sonuç, günlük bazda virüsler tarafından tahrip hacim başına hücre bolluğu. Sonra, söz konusu sistem için virüs kaynaklı ölüm ile sonuçlanan bakteriyel bolluk ayakta stok değeri parçalanmış mikropların ayrılabilir: mevcut tahminleri aralığı az yüzde neredeyse tüm nüfusu ve sık sık söz konusu sistem diğer faktörlere bağlıdır. (Wilhelm &amp; Matteson 2008). Toplam mortalite yüzdesi belirlemek için bu sayı genellikle iki (% 50 hücrelerin yeniden gidin ve hücrelerin% 50, Weinbauer 2004 kayıp olduğunu varsayım çalışan) ile çarpılır. Haline gelmiştir besin ve iz element biyoyararlanım (örneğin, N, P, Fe) birincil üretkenlik oranı sınırı, ve karbon akı olarak, sucul sistemleri aracılığıyla, ve bu süreç içinde, virüs kaynaklı mikrobiyal ölüm rolü anlaşılması için verilen deniz jeokimyacılar ilgi. Çeşitli tahminlere şimdi (2009 2008, Higgins ve ark. Rowe ve ark) virüsler günlük bazda önemli bir besin elementi konsantrasyonları serbest su sütunu geri öneririz var ve bu unsurların hızla mikrobiyal toplum tarafından asimile (Poorvin et diğerleri, 2004, Mioni ve ark 2005). Miktarda besin hücre başına (başlar &quot;kotaları) tarafından tahrip hücrelerinin sayısı çarpılarak ortamda besin akı oranı tespit edilebilir. Bu bilgiler, mikrobiyal gıda ağları su sistemleri arasında nasıl işlediğini anlamak için kritik bir bileşen sağlayabilir. Devam eden gelişmeler: Şimdiki çabalar, bir dizi araştırma grupları gibi belirli organizmaların virüs aktivitesi tarafından nasıl etkilendiğini belirlemek için, toplum içinde belirli virüsleri numaralandırılamadı Yukarıda ifade edilen stratejinin uyarlanması ve içerir. Bunu yapmak için araştırmacılar, toplam virüs toplumun tahminlerine paralel olarak belirli virüslere gruplar veya aileler bolluğu tahmin etmek için kantitatif polimeraz zincir reaksiyonu (qPCR) kullanın. Sonuçlar daha sonra doğrudan belirli plankton gruplar için virüs ölüm, besin ciro, vb tahminleri sağlamak için uygulanır. Bu yeni ve güçlü bir yaklaşım, önümüzdeki yıllarda araştırmacılar, virüslerin ekoloji ile ilgili süreçlere çok daha derin kazmak ve ilk kez, laboratuar sistemleri kısıtlamaları ötesinde belirli virüs host toplulukların etkileşimi ölçmek için izin verecektir.

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>2.5% p-phenylenediamine</td>
<td>&nbsp;</td>
<td>Acros</td>
<td>130575000</td>
<td>Stock for Antifade</td>
</tr>
<tr>
<td>Amicon Proflux M12 system</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>N/A</td>
<td>Any ultrafiltration device may be used for this step</td>
</tr>
<tr>
<td>SYBR Green I nucleic acid gel stain</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>S-7563</td>
<td>&nbsp;</td>
<tr>
<td>Helicon S10 30kDa Filter</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>CDUF010LT</td>
<td>&nbsp;</td>
<tr>
<td>Pelicon XL filters 0.22 &mu;m</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>PXGVPPC50</td>
<td>&nbsp;</td>
<tr>
<td>GE 0.2 &mu;m PCTE membrane filters (47mm)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>09-732-35</td>
<td>&nbsp;</td>
<tr>
<td>Millipore Labscale Tangential Flow Filtration System</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>XX42LSS11</td>
<td>Other TFF systems may be used for this step</td>
</tr>
<tr>
<td>0.45 &mu;m Micronstep, Cellulosic, white plain filters (25mm)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>E04WP02500</td>
<td>&nbsp;</td>
<tr>
<td>GE Whatman 0.02 &mu;m Anodisc filters (25mm)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>68-09-6002</td>
<td>&nbsp;</td>
<tr>
<td>Leica DMRXA microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Any epifluorescence microscope with a blue filter set may be used</td>
</tr>
<tr>
<td>20L polycarbonate carboys</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Glycerol</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP229-4</td>
<td>&nbsp;</td>
<tr>
<td>PBS (0.05 M Na2HPO4, 0.85% NaCl, pH 7.5)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP329-500, S640-500</td>
<td>&nbsp;</td>
<tr>
<td>50% Glutaraldehyde </td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>G151-1</td>
<td>&nbsp;</td>
<tr>
<td>Corning 2 mL Cryovials-External Thread polypropylene</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>09-761-71</td>
<td>Any cryovials may be used</td>
</tr>
<tr>
<td>Corning 5 mL Cryovials-External Thread polypropylene</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>09-761-74</td>
<td>Any cryovials may be used</td>
</tr>
<tr>
<td> 85% H3PO4</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>A242-212</td>
<td>Spiral Membrane storage</td>
</tr>
<tr>
<td>NaOH pellets</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>S318-3</td>
<td>M12 cleaning</td>
</tr>
<tr>
<td>Graduated Cylinders</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Isopore 0.8-&mu;m pore-size membrane filter (142mm)</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>ATTP14250</td>
<td>&nbsp;</td>
<tr>
<td>Millipore Stainless Steel Pressure Filter Holder (142mm)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>YY30 090 00</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

estimating virus production rates in aquatic systems

Virüsler, deniz ve tatlı su sistemlerinin yaygın bileşenleri ve mikrobiyal ölüm önemli ajanları olduğu bilinmektedir. Sonra mikrobiyal toplum yapısı ve işlevi daha iyi modelleri geliştirmek gibi virüsler sucul biyojeokimyasal döngülerini değiştirmeye nasıl çalıştığını anlayışımızı ilerletmek gibi bu sürecin niceliksel tahminler geliştirilmesi kritik öneme sahiptir. Virüs azaltma tekniği araştırmacıları virüs parçacıkları endemik mikrobiyal topluluk yayımlanan hızını tahmin etmek sağlar. Mikrobiyal topluluk yakın bir ortam konsantrasyon korunurken Kısacası, ücretsiz (ekstraselüler) virüslerin bolluğu bir örnek azalır. Mikrobiyal topluluk, daha sonra ücretsiz virüs olmaması ve virüsler kantitatif (topluluk zaten bulaşmış üyelerinin lizis yoluyla) örnek reoccur Epifloresans mikroskobu ile belirlenebilir veya belirli virüslere durumda, hangi oranı inkübe PCR. Bu oranlar daha sonra virüs aracılı hücre erimesi nedeniyle mikrobiyal ölüm oranını tahmin etmek için kullanılabilir.

Watch this Scientific Journal Video about Estimating Virus Production Rates in Aquatic Systems at JoVE.com

Estimating Virus Production Rates in Aquatic Systems

Deniz ve tatlı su sistemlerinin virüslerin ciro oranı bir azalma ve yeniden oluşmaları tekniği ile tahmin edilebilir. Veriler araştırmacılar, su sistemlerinde virüs aracılı mikrobiyal ölüm oranları anlaması için izin verir.

Su Sistemleri Virüs Üretim Oranlar tahmin

Viruses are pervasive components of marine and freshwater systems, and are known to be significant agents of microbial mortality. Developing quantitative ...

estimating-virus-production-rates-in-aquatic-systems

Research

JoVE Journal

Immunology and Infection

12.6K Görüntüleme.  University of Tennessee. Deniz ve tatlı su sistemlerinin virüslerin ciro oranı bir azalma ve yeniden oluşmaları tekniği ile tahmin edilebilir. Veriler araştırmacılar, su sistemlerinde virüs aracılı mikrobiyal ölüm oranları anlaması için izin verir.

Video: Estimating Virus Production Rates in Aquatic Systems

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

Alexa Fluor Etiketleme Dang Virüs görselleştirme

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

İmmünoloji ve Enfeksiyon

Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 which regulate the expression of non-structural (NS1 and NS2) and capsid proteins (VP1 and VP2) respectively1. They attract high interest as anticancer agents for their oncolytic and oncosuppressive abilities while being non-pathogenic for humans2. NS1 is the major effector of viral cytotoxicity3. In order to further enhance their natural antineoplastic activities, derivatives from these vectors have been generated by replacing the gene encoding for the capsid proteins with a therapeutic transgene (e.g. a cytotoxic polypeptide, cytokine, chemokine, tumour suppressor gene etc.)4. The recombinant parvoviruses (recPVs) vector retains the NS1/2 coding sequences and the PV genome telomeres which are necessary for viral DNA amplification and packaging. Production of recPVs occurs only in the producer cells (generally HEK293T), by co-transfecting the cells with a second vector (pCMV-VP) expressing the gene encoding for the VP proteins (Fig. 1)4. The recPV vectors generated in this way are replication defective. Although recPVs proved to possess enhanced oncotoxic activities with respect to the parental viruses from which they have been generated, their production remains a major challenge and strongly hampers the use of these agents in anti-cancer clinical applications.
We found that introduction of an Ad-5 derived vector containing the E2a, E4(orf6) and the VA RNA genes (e.g. pXX6 plasmid) into HEK293T improved the production of recPVs by more than 10 fold in comparison to other protocols in use. Based on this finding, we have constructed a novel Ad-VP-helper that contains the genomic adenoviral elements necessary to enhance recPVs production as well as the parvovirus VP gene unit5. The use of Ad-VP-helper, allows production of rec-PVs using a protocol that relies entirely on viral infection steps (as opposed to plasmid transfection), making possible the use of cell lines that are difficult to transfect (e.g. NB324K) (Fig. 2). We present a method that greatly improves the amount of recombinant virus produced, reducing both the production time and costs, without affecting the quality of the final product5. In addition, large scale production of recPV (in suspension cells and bioreactors) is now conceivable.

Here we describe a protocol based only on cell infection, which improves the efficiency of recombinant parvovirus production by more than 100 fold in comparison to other protocols in use. This protocol relies on the use of a novel adenovirus 5-based helper containing the parvovirus VP transcription unit (Ad-VP).

Adenovirüs kaynaklı Sistemleri Yardımı ile verimli Rekombinant Parvovirus Üretim

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 ...

In Vivo Imaging Systems (IVIS) Detection of a Neuro-Invasive Encephalitic Virus

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by Russel and Burch in Hume's 3Rs (replacement, reduction, refinement), the utilization of animal models in scientific research is under constant pressure to identify new methodologies to reduce animal usage while improving scientific accuracy and speed. A major challenge to Hume's principals however, is how to ensure the studies are statistically accurate while reducing animal disease morbidity and overall numbers. Vaccine efficacy studies currently require a large number of animals in order to be considered statistically significant and often result in high morbidity and mortality endpoints for identification of immune protection. We utilized in vivo imaging systems (IVIS) in conjunction with a firefly bioluminescent enzyme to progressively track the invasion of the central nervous system (CNS) by an encephalitic virus in a murine model. Typically, the disease progresses relatively slowly, however virus replication is rapid, especially within the CNS, and can lead to an often, lethal outcome. Following intranasal infection of the mice with TC83-Luc, an attenuated Venezuelan equine encephalitis virus strain modified to expresses a luciferase gene; we are able to visualize virus replication within the brain at least three days before the development of clinical disease symptoms. Utilizing CNS invasion as a key encephalitic disease development endpoint we are able to quickly identify therapeutic and vaccine protection against TC83-Luc infection before clinical symptoms develop. With IVIS technology we are able to demonstrate the rapid and accurate testing of drug therapeutics and vaccines while reducing animal numbers and morbidity.

In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus

Utilizing luciferase and in vivo imaging systems (IVIS) as a novel means to identify disease endpoints before clinical developments occur. IVIS has allowed us to visualize in real time the invasion of encephalitic viruses over multiple days, providing a more accurate disease model for future study. It has also allowed us to identify the potential protective features of antivirals and vaccines faster than currently utilized animal models. The capability to utilize individual animals over multiple time points ensures reduced animal requirements, costs, and overall morbidity to the animals utilized ensuring a more humane and more scientific means of disease study.

Bir Nöro-İnvazif encephalitic Virüs Vivo Görüntüleme Sistemleri (IVIS) Algılama

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

Embriyone Tavuk Yumurta Grip Virüsü Yayılım

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Nötrofil İzolasyon ve Analiz Terapötik Ajanların için Lenfoma Hücre Duyarlılık kendi Rolü belirleme

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

Basit ve Etkin Yaklaşım Mutant Vaccinia Virüs Vektörler Construct

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

Bir gen ya da ilaç terapisinde kullanım için, HIV-1 üretim yapan RNA&#39;lar etkinlik ve toksisite Değerlendirilmesi

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

Tam kan örneklerinde virüs tespiti için hızlı, güvenli ve basit el ile başucu nükleik asit çıkarma

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying gene expression related to biological processes of interest. These innovations not only allow researchers to observe expression from the mRNA sequences that code for genes that effect cellular function, but also the non-coding RNA (ncRNA) molecules that remain untranslated, but still have regulatory functions. Although researchers have the ability to observe both mRNA and ncRNA expression, it has been customary for a study to focus on one or the other. However, when studies are interested in both mRNA and ncRNA expression, many times they use separate samples to examine either coding or non-coding RNAs due to the difference in library preparations. This can lead to the need for more samples which can increase time, consumables, and animal stress. Additionally, it may cause researchers to decide to prepare samples for only one analysis, usually the mRNA, limiting the number of biological questions that can be investigated. However, ncRNAs span multiple classes with regulatory roles that effect mRNA expression. Because ncRNA are important to fundamental biologic processes and disorder of these processes in during infection, they may, therefore, make attractive targets for therapeutics. This manuscript demonstrates a modified protocol for the generation mRNA and non-coding RNA expression libraries, including viral RNA, from a single sample of whole blood. Optimization of this protocol, improved RNA purity, increased ligation for recovery of methylated RNAs, and omitted&#160;size selection, to allow capture of more RNA species.

Here, we present a protocol optimized for the processing of coding (mRNA) and non-coding&#160;(ncRNA) globin reduced RNA-seq libraries from a single whole blood sample.

Kodlama ve RNA sınıfları kodlamayan teşhis kodu içinde domuz tam kan ifade.

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...

Measurement of Specific Mycobacterial Mistranslation Rates with Gain-of-function Reporter Systems

The translation of genes into proteins is prone to errors. Although the average rate of translational error in model systems is estimated to be 1/10,000 per codon, the actual error rates vary widely, depending on the species, environment, and codons being studied. We have previously shown that mycobacteria use a two-step pathway for the generation of aminoacylated glutamine and asparagine tRNAs and that this is specifically associated with relatively high error rates due to the modulation of mistranslation rates by an essential component of the pathway, the amidotransferase GatCAB. We modified a previously employed Renilla-Firefly dual-luciferase system that had been used to measure mistranslation rates in Escherichia coli for use in mycobacteria to measure specific mistranslation rates of glutamate at glutamine codons and aspartate for asparagine codons. Although this reporter system was suitable for the accurate estimation of specific error rates, lack of sensitivity and requirements for excessive manipulation steps made it unsuitable for high-throughput applications. Therefore, we developed a second gain-of-function reporter system, using Nluc luciferase and green fluorescent protein (GFP), which is more amenable to medium/high-throughput settings. We used this system to identify kasugamycin as a small molecule that can decrease mycobacterial mistranslation. Although the reporters that we describe here have been used to measure specific types of mycobacterial mistranslation, they may be modified to measure other types of mistranslation in a number of model systems.

In this article, we present two complementary methods to measure specific rates of translational error and mistranslation in the model mycobacterium, Mycobacterium smegmatis, using gain-of-function reporter systems. The methods can be employed to measure accurate error rates in low throughput or relative error rates in a more high-throughput setting.

Fonksiyon kazancı Reporter sistemleri ile spesifik Mikobakteriyel güvenirlik oranlarının ölçülmesi

The translation of genes into proteins is prone to errors. Although the average rate of translational error in model systems is estimated to be 1/10,000 ...