A novel mechanism is proposed for possible involvement of the elaborated pseudoknots in the HIV1 PRF event.Programmed −1 ribosomal frameshifting (PRF) and stop codon readthrough are two translational recoding mechanisms utilized by some RNA viruses to express their structural and enzymatic proteins at a defined ratio. Efficient recoding usually requires an RNA pseudoknot located several nucleotides downstream from the recoding site. To assess the strategic importance of the recoding pseudoknots, we have carried out a large scale genome-wide analysis in which we used an in-house developed program to detect all possible H-type pseudoknots within the genomic mRNAs of 81 animal viruses. Pseudoknots are detected downstream from ~85% of the recoding sites, including many previously unknown pseudoknots. ~78% of the recoding pseudoknots are the most stable pseudoknot within the viral genomes. However, they are not as strong as some designed pseudoknots that exhibit roadblocking effect on the translating ribosome. Strong roadblocking pseudoknots are not detected within the viral genomes. These results indicate that the decoding pseudoknots have evolved to possess optimal stability for efficient recoding. We also found that the sequence at the  During the translation process, ribosomes are capable of performing some nonstandard decoding events which provided that appropriate signals are present in the mRNA being translated. These unusual events are referred to as “recoding” , 2. Two gag-pol polyprotein from the overlapping gag and pol open reading frames from a single translation initiation codon of the 5′gag reading frame [gag and pol gene, respectively. For efficient −1 frameshifting to happen, two cis-acting elements programmed in the overlapping region of the mRNA are often required to signal the translating ribosomes to shift backward by one nucleotide. The first element is a heptanucleotide stretch termed “slippery sequence” with a typical composition of X XXY YYZ . Although the slippery sequence is the site of action where the ribosomes actually shift to the −1 frame, this element alone is not sufficient to cause efficient shifting. A secondary signal called “stimulator” is usually required in the form of an RNA structure downstream from the frameshift site, separated by a spacer region . In some cases, the stimulator is a conventional stem-loop structure, but most often it is a pseudoknot, which is a structural motif of RNA formed when a stretch of nucleotides within a loop region in a secondary structure basepairs with residues outside that loop [The discovery of the −1 PRF mechanism was made by Atkins and coworkers , and theng frame . In −1 fhat loop –10 , a pseudoknot located several nucleotides 3′ to the UAG termination codon of the gag gene was found to be required [gag-pol region of MuLV and the other viruses of the readthrough retrovirus group imply that the other readthrough retroviruses may use a similar pseudoknot structure to stimulate the stop codon readthrough as well [ The involvement of RNA pseudoknots in stop codon readthrough has also been established. In the required , 21. Str as well , 23.  The vast majority of the established frameshift- or readthrough-stimulating pseudoknots belong to the so-called H (hairpin)-type pseudoknots, in which a stretch of nucleotides within a hairpin loop basepairs with a complementary region outside of the hairpin see . gag-pro junctions from the mouse mammary tumor virus (MMTV) (24–26) and the simian retrovirus-1 (SRV-1) [ The structures of many −1 frameshift stimulating pseudoknots have been determined by NMR or X-ray crystallography, including those at the s (MMTV) –26 and t (SRV-1) , the P1- (SRV-1) , the pea (SRV-1) , the pot (SRV-1) , and the (SRV-1) . In the  It is known that some mRNA structures, especially pseudoknots, can cause the translating ribosome to pause upstream to such structures –34. In aG) between folded and unfolded pseudoknots. The requirement for extra energy input may explain why the pseudoknot is more resistant to unfolding by optical tweezers (presumably by ribosomes as well). The mechanical strength of pseudoknots was further correlated with the frameshift stimulating ability of the pseudoknots [ Single molecule studies using optical tweezers to pull pseudoknots apart –39 showeudoknots , 38. By  A more recent study confirmed the ribosomal roadblocking effect of strong pseudoknots . By inve Given these recent progresses on the mechanisms of frameshifting, it would be interesting to assess the uniqueness of the frameshift stimulating pseudoknots in the viral genomic RNAs. How many other potential pseudoknots are present in the viral mRNAs? How do the other pseudoknots compare to the frameshift stimulating pseudoknots in terms of thermodynamic stability and mechanical strength? Are there strong roadblocking pseudoknots in the viral genomic mRNAs? To address these questions, we have developed a computer program capable of identifying potential H-type pseudoknots in any given mRNA sequence and ranking the identified pseudoknots according to the relative strength of their helical stems. Using the program, we have analyzed the full-length genomic mRNAs of 81 animal viruses that are known or expected to use −1 frameshifting or readthrough as a decoding mechanism for protein expression. A computer program has been developed to identify all putative H-type pseudoknots within any given RNA sequence . G37°) calculation for the two helical stems S1 and S2. In calculating the free energy, the Turner's nearest-neighbor parameters are used [ In order to compare the relative thermodynamic stability and mechanical strength of the identified pseudoknots within a given mRNA sequence, we implemented free energy (Δare used . If L3 =http://dx.doi.org/10.1155/2013/984028).  The output file of the program contains information about whether pseudoknots are found and how many are found; the detected pseudoknots are then listed in the order of calculated free energy of the stems. For each of the detected pseudoknots, the following information is given: lengths of S1, S2, L1, L2, and L3; free energy value of the stems; size and location of the pseudoknot. A schematic diagram is then drawn showing the actual pseudoknot forming sequence and base pairing of the two stems; a sequence of 20 nucleotides immediately 5′ - to the pseudoknot is also shown because frameshift or readthrough pseudoknots usually appear several nucleotides downstream of the frameshift or readthrough sites , Lelystad virus , Lactate dehydrogenase-elevating virus , Porcine reproductive and respiratory syndrome virus , and Simian hemorrhagic fever virus . Coronaviridae family: Transmissible gastroenteritis virus , Bovine coronavirus , Human coronavirus 229E , Human coronavirus OC43 , Porcine epidemic diarrhea virus , Human coronavirus HKU1 , Murine hepatitis coronavirus , SARS coronavirus , avian infectious bronchitis coronavirus , Bovine torovirus (Breda virus) , and Equine torovirus (Berne Virus) . Astroviridae family: Chicken astrovirus , Turkey astrovirus , Human astrovirus , Mink astrovirus , and Ovine astrovirus . Flaviviridae family: Japanese encephalitis virus , Murray Valley encephalitis virus-Alfuy , Murray Valley encephalitis virus , Usutu virus , West Nile virus-Kunjin , West Nile virus H442 , West Nile virus . Retroviridae family: avian leukosis virus HPRS-103 ; Rous sarcoma virus ; jaagsiekte sheep retrovirus ; Mason-Pfizer monkey virus ; simian retroviruses type-1  and type-2 ; mouse mammary tumor virus ; squirrel monkey retrovirus ; human endogenous retrovirus K10 ; three intracisternal A particle (IAP) genetic elements from Chinese hamster , Syrian hamster , and mouse ; bovine immunodeficiency virus ; human T-cell leukemia virus type-I  and type-II ; simian T-cell leukemia virus type-I ; Walleye dermal sarcoma virus AF033822; baboon endogenous virus ; Feline leukemia virus ; gibbon ape leukemia virus ; Moloney murine sarcoma virus ; Moloney murine leukemia virus ; Friend murine leukemia virus ; Akv murine leukemia virus ; bovine leukemia virus ; Jembrana disease virus ; Ovine lentivirus ; South African Ovine Maedi Visna virus ; Caprine Arthritis Encephalitis virus ; feline immunodeficiency virus ; equine infectious anaemia virus ; puma lentivirus ; human immunodifeciency virus type 1 strain ; human immunodeficiency virus type 2 ; simian immunodeficiency virus . Togaviridae family: Aura virus ; Barmah Forest virus ; Chikungunya virus ; Eastern equine encephalitis virus , Fort Morgan virus ; Getah virus ; Highlands J virus ; Mayaro virus ; Middelburg virus ; Ndumu virus ; O'nyong-nyong virus ; Ross River virus ; Salmon pancreas disease virus ; Seal louse virus ; Semliki Forest virus ; Sindbis virus ; Venezuelan equine encephalitis virus ; Western equine encephalitis virus ; Whataroa virus . Most of the viruses and their sequence sources investigated in this study are taken from Recode V2.0: Database of translational recoding events . The namIn our study, a total of 81 full-length genomic RNA sequences of animal viruses were analyzed for the presence of potential H-type pseudoknots. To facilitate the analysis of such a large number of full-length sequences, in house-developed computer program is used, which is capable of identifying H-type pseudoknots efficiently and reliably. In brief, the program identifies pseudoknots by scanning through the input RNA sequence and testing every possible combination of stem and loop  lengths within the predefined ranges to see whether two helical stems can form simultaneously in a linear sequential topology as shown in  In our pseudoknot search, we set the default ranges for stem and loop lengths as follows: stem1 (S1) and stem2 (S2) both have from 5 to 20 base pairs, loop1 (L1) has from 1 to 10 nucleotides, loop2 (L2) has from 3 to 50 nucleotides, and loop3 (L3) has from 0 (L3 is absent) to 10 nucleotides. These default ranges are very generous because most established H-type pseudoknots have stem and loop lengths that fall within these ranges.  To evaluate the relative strength of the identified pseudoknots in a given viral genomic RNA sequence, the free energy of the two stems for each of the identified pseudoknots was calculated, based on the Turner's nearest-neighbor parameters. The pseudoknots were ranked according to the calculated free energies. For a bioinformatic investigation, this calculated free energy represents the best way to evaluate the relative stabilities of the putative H-type pseudoknots within a given viral genome. The free energy value is also used as a criterion for the search; only those pseudoknots with a free energy value lower than −18 kcal/mol are kept for further analysis.gag-pro junction [ In a typical search, tens of potential pseudoknots were identified within the full-lengths genomic viral mRNAs. For example, in the full-length genomic RNA of simian retroviruses type-1  that has 8173 nucleotides, 50 potential pseudoknots were identified using the default stem and loop ranges for pseudoknot formation. Some of these potential pseudoknots have overlapped pseudoknot-forming sequences; that is, two or more potential pseudoknots are mutually excluded and cannot exit at the same time. After eliminating overlapped pseudoknots with higher free energy, the number of potential pseudoknots in the SRV-1 genomic mRNA decreases to 31. Of course, it is possible that some of the detected pseudoknots may not really exist. Among the 31 detected pseudoknots, the established −1 frameshift stimulating pseudoknot at the junction , 43 is i While pseudoknots were detected shortly downstream from the frame-shift or read-through sites in most of the viral sequences using the default ranges of stem and loop lengths, the default search did miss some known cases, such as the frameshift stimulator pseudoknot in human coronavirus 229E that has a 164 nt L2. For such cases, the ranges of stem and loop lengths were increased accordingly for another round of search. At the end, possible pseudoknots were identified shortly downstream from the frame-shift or read-through sites in 69 full-length viral genomic mRNA sequences (85% of the 81 sequences).  As documented in Recode V2.0: database of translational recoding events , a largeThe Arteriviridae Family. −1 frameshift stimulating pseudoknots identified in this family of viruses are pretty much identical to those shown in the Recode database , also has a potential frameshift stimulating pseudoknot, which seems to be different from those pseudoknots in EAV, LV, LDV, and PRRSV. The potential frameshift stimulating pseudoknot in SHFV ranks 78th among all of the potential pseudoknots identified within the full-length genomic RNAs. The first ranked potential pseudoknot has a calculated stem free energy of −31.7 kcal/mol.The Coronaviridae Family. PRF stimulating pseudoknots identified in this family of viruses at the ORF1a and ORF1b overlapping region are basically the same as those shown in the Recode database and a number of previous studies [ studies , 47–50.  The HCV229E frameshifting pseudoknot has a long (164 nt) L2. It was found that a short stretch of nucleotides at the 3′-end of L2 participated in the formation of an extra helical stem required for efficient frameshifting in HCV229E . The extThe frameshift stimulator pseudoknot in PEDV is different from those pseudoknots in other viruses of this family. Most noticeably, the length of S1 is much shorter (5 bp versus 11–14 bp). Correspondingly, the pseudoknots are less stable. It ranks beyond 100th among all the potential pseudoknots within the gemones. The 1st ranked putative pseudoknot in this virus has a calculated free energy of −38.7 kcal/mol. The Astroviridae Family. There are five astroviruses  in the Recode database. All viruses use the same slippery sequence A AAA AAC. According to the Recode database, human astrovirus and chicken astrovirus use a pseudoknot and a stem-loop as the frameshift stimulator, respectively, while there is no information on the other three viruses. However, we detected potential pseudoknots 6–8 nt downstream from the slippery sequences in ovine, turkey and chicken astroviruses. The detected pseudoknots have comparable stem and loop lengths, as well as stem free energies. They rank 2nd, 10th, and 16th among all the potential pseudoknots within the gemones.The Flaviviridae Family. Pseudoknots detected in this family are identical to those documented in the Recode database. Two slippery sequences are used by these viruses: C CCU UUU and U CCU UUU. The pseudoknots are very similar to each other in terms of the lengths of the stems & loops and stability. In all but one of the viruses, the putative frameshift stimulating pseudoknot rank 1st among all potential pseudoknots within the genome. The frameshift stimulating pseudoknot in Murray Valley encephalitis virus ranks 2nd. The 1st ranked pseudoknot has a calculated stem free energy of −38.9 kcal/mol, which is slightly lower than the frameshift stimulating pseudoknot.The Retroviridae Family. Viruses in the retroviridae family belong to several genera: alpha-, beta-, gamma-, delta-, or epsilon-retroviruses and lentivirus. These retroviruses utilize three different mechanisms to express their gag, pro and pol genes from a single gag-pro-pol translational unit: (1) in-frame readthrough of the gag termination codon (gamma- and epsilon-retroviruses); (2) single frameshift event at the gag-pol junction to express the pol gene ; (3) double frameshift events at the gag-pro and pro-pol junctions to express the pro and pol genes (beta- and delta- retroviruses). gag-pol frameshift junction and both have a pseudoknot. The pseudoknot in RSV as a frameshift stimulator has been established [ The two alpharetroviruses Avian leukosis virus (ALV) and Rous sarcoma virus (RSV) have very similar sequences at the ablished . The ALVablished , 52. Howpro and pol genes. gag-pro frameshifting  and L2 (ranging from 15 to 52 nt); the total numbers of basepairs in the two stems are also larger (ranging from 14 to 20 bp). The functional importance of the gag-pro frameshift pseudoknots in the betaretroviruses has been well established [pro-pol frameshifting has not been established. No pseudoknot was detected downstream from the ablished , 53–55. pro and pol genes. The secondary RNA structures downstream of the gag-pro frameshift sites in this group of retroviruses were generally believed to be simple stem-loops [gag-pro frameshift site in all viruses but BLV (not shown). However, credibility of the detected pseudoknots is questionable due to the lack of appropriate spacer between the slippery sequence and the pseudoknot. In view of the fact that the previously reported stem-loop structures seem to be conserved in all four viruses, we increased the upper limit for L2 to 85 nt and performed another search. Interestingly, potential pseudoknots with decent stability were detected downstream from the gag-pro frameshift site in all four viruses  lentiviruses: simian immunodeficiency viruses (SIV) and human immunodeficiency viruses type-1 and type-2 (HIV-1 and HIV-2) in the Recode database. The database gives only one representative sequence for each of these viruses. These are the particular sequences we investigate in this study. No potential pseudoknot was detected downstream from the gag-pol frameshift site harbours two potential pseudoknots that are mutually excluded  type [ In HIV-1 (strain HXB2), the sequence downstream from the excluded . One of excluded . The 1stexcluded . The two 1) type , 61  were investigated. For three of these viruses , the Recode database predicts a pseudoknot structure downstream from the frameshift site. For the other viruses, the Recode database either predicts a simple stem-loop structure downstream from the frameshift site or makes no prediction. Using our pseudoknot search method, we detected putative frameshift stimulator pseudoknots in twelve viruses, including MIDV, NDUV and SESV . All but one of these pseudoknots were detected by our program. The missed case is in human astrovirus, in which the predicted framshift stimulator pseudoknot by the Recode database is very weak. Importantly, our approach of pseudoknot detection was not restricted to a limited sequence window downstream from the known frameshift or readthrough recoding sites. Instead, the program detects all possible pseudoknots within the full-length viral genomic mRNAs. The effectiveness and reliability of our approach are proven by the fact that almost all of the previously documented frameshift or readthrough stimulator pseudoknots are detected. gag-pro and pro-pol sites in the same virus are counted as two different sites). Some of these detected pseudoknots may not actually exist. However, the high percentage of possible pseudoknots detected downstream from the strategically important frameshift or readthrough sites still overwhelmingly proves that pseudoknots are the most common stimulators for efficient −1 ribosomal frameshifting and readthrough.  Interestingly, we also detected quite a number of putative frameshift stimulator pseudoknots that were not known before. Overall, potential pseudoknots were detected downstream from most (~90%) of the established or putative frameshift or readthrough sites  belonged to a structurally related pseudoknot family known as CPK-1, standing for common pseudoknot motif 1 , 61. A tSARS pseudoknots) which would make the pseudoknots even more versatile in fine-tuning the frameshifting.Consistent with this theory, it was found in several different systems that frameshifting efficiency was generally more sensitive to mutations introduced to S1 & L2, and the junction, while mutations to S2 & L1 showed less effect on frameshifting efficiency, as long as integrity and stability of the pseudoknot-forming interaction were maintained , 63–66.  Although CPK-1 type pseudoknots occur most frequently among the detected pseudoknots, other types of pseudoknots are also observed. These pseudoknots sample a wide range of stem and loop sizes, as well as the presence or absence of an intervening sequence (L3). Given the wide range of sequences studied, it is not particularly surprising to observe all these variations associated with the detected pseudoknots. Since different viruses  may have different requirements for certain level of frameshifting efficiency, variations in the frameshifting pseudoknots, as well as the slippery sequences and spacers, may be necessary for the fine-tuning of the frameshifting efficiency to meet the specific need of different viruses. gag-pol frameshift site was originally proposed to be a simple stem-loop [in vivo  [gag-pol frameshift site was shown to be required for stimulating a higher frameshifting efficiency than that in group M [ The frameshift stimulating secondary structure downstream of the HIV-1 group M (which includes the strain HXB2 investigated in this study) tem-loop , which wn cells) . It was n cells)  or an exn cells) . In HIV- group M . gag-pol junction of both HIV-1 and HIV-2. In HIV-1 (HXB2), two mutually excluded potential pseudoknots were detected downstream from the slippery sequence . These pseudoknots can significantly slow down the translating ribosome when the pseudoknots are being unwound by the ribosome. When the ribosome scans through the unwound mRNA sequence and is approaching the slippery sequence, the top pseudoknot is fully unwound and the stem1 of the bottom pseudoknot should also be disrupted. Stem2 of the bottom pseudoknot remains intact. The six basepairs of this stem2 are actually the same as the first six basepairs in stem1 of the mutually excluded pseudoknot  which are presumably important for the fine-tuning of framshift or readthrough stimulating ability of the pseudoknots. Fourth, some HIV1 viruses may utilize a novel mechanism that involves three pseudoknots to regulate the frameshift efficiency at the In a computer-generated output (figure below), the sequence elements of the detected pseudoknot  and a sequence of 20 nt preceding the pseudoknot are shown in three lines. The first line is for L2 and L1. The second line is for S1-3' and S2-5' sequences. The third line is for the 20 nt preceding sequence (which may contain the slippery sequence and spacer region), S1-5', and S2-3' sequences. Note that the 5'→3' orientation of the sequence in the second line runs from the right to the left, which is opposite to the sequences in the other two lines. The free energy (∆Go37) values for the two stems S1 and S2 are calculated and indicated. The output also indicates the starting/ending positions of the pseudoknot-forming sequence, the number of nucleotides in the stems/loops  of the pseudoknot. The detected pseudoknots in a viral genome are ranked by the calculated free energy values.In the sample output, some pseudoknots have their pseudoknot-forming sequences overlapped (such as the first and second ranked pseudoknots), meaning that the pseudoknots cannot exist simultaneously. The overlapped pseudoknots with a higher free energy are indicated by a line through the middle of the selected text.Click here for additional data file.


To the Editor: Several exanthematic vaccinia virus (VACV) outbreaks have affected dairy cattle and rural workers in Brazil and Asia, and have caused economic losses and affected health services (–Orthopoxvirus (OPV), exhibits serologic cross-reactivity with other OPV species and was used during the smallpox eradication campaign  who worked on a farm  was hospitalized. He had painful lesions on the hands, high fever, lymphadenopathy, malaise, and fainting episodes. This patient reported recent contact with sick animals on the farm during milking.At the same time, a 57-year-old man (patient 2), the owner of the farm, had a lesion on the right hand. This infection was also related to occupational exposure. Some days after the appearance of the hand lesion, this patient presumably inoculated himself at the site of an abrasion he had recently received on his nose. This resulted in development of a large and painful lesion. This patient reported milking cows daily. He had been vaccinated against smallpox before 1977.g for 3 min. The supernatants were used for molecular diagnosis and virus isolation , and the A26L gene, which encodes A-type inclusion (ATI) protein. Samples from the 2 patients were positive for vgf and ATI , amphotericin B (4 μg/mL), and gentamicin (100 μg/mL); homogenized, and centrifuged at 2,000 × vgf and ATI by PCR.To assess the risk for virus spread in indoor environments, we collected swab specimens from several objects, including doorknobs, bathroom surfaces, and the pillow of patient 2. The pillow was positive for ,,To isolate the virus, we infected monolayers of BSC-40 cells cultured in a 6-well plate with sample supernatants and incubated the cells at 37°C for 72 h or until a cytopathic effect was detected  method and manually aligned by using MEGA version 4.0 . VACV molecular signatures of 18-nt and 12-nt deletions were observed in the A56R and A26L genes, respectively. Phylogenetic trees . Sequences were aligned with published OPV sequences in GenBank by using the ClustalW (ic trees , which w,We isolated VACV from an indoor environment during a bovine vaccinia outbreak. VACV infections have been frequently associated with occupational activities, primarily direct contact with sick animals (,Human-to-human transmission has been suggested to have occurred in some bovine vaccinia outbreaks in Brazil, and nosocomial infection has been reported Asia (


P = 0.0162), high serum α-fetoprotein level (P = 0.0001), high tumor grade (P = 0.0029), high tumor stage (P = 0.0319), the presence of the p53 mutation (P = 0.0032), and the absence of the β-catenin mutation (P = 0.0489). Patients with HCC tumors that expressed S100P were more likely to have early tumor recurrence (ETR) (P = 0.0189) and lower 5-year survival (P = 0.0023). The multivariate analysis confirmed that S100P expression was an independent prognostic factor in HCC. The combinatorial analysis showed an additive unfavorable prognostic interaction between S100P expression and the p53 mutation. In contrast, the β-catenin mutation was associated with better prognosis in both S100P-positive and -negative HCCs. Furthermore, S100P expression was a predictor of survival in HCC patients with high tumor stage or ETR . Our study indicates the expression of the S100P protein is a novel independent predictor for poor prognosis in HCC, and it is also an unfavorable prognostic predictor in HCC patients with high tumor stage or ETR.The calcium-binding protein S100P is expressed in a variety of human cancer cells and is important in cancer cell growth and invasion. Using differential display, we found S100P is overexpressed in human hepatocellular carcinoma (HCC). We examined the expression of 305 unifocal, primary HCC tumors using immunohistochemistry. The S100P protein was expressed in 173 of the 305 (56.7%) HCC tumors. The expression of S100P correlated with female sex ( Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, particularly in Taiwan, southern China, Southeast Asia, and sub-Saharan Africa, and the incidence of HCC is increasing in Western countries Early tumor recurrences (ETRs) arise primarily from intrahepatic metastases, and a significantly poorer prognosis S100P, which is preferentially expressed in tumors.One way to elucidate the pathogenesis of cancers is to identify the genes that are up- and down-regulated during the disease course. Methods for detecting such altered expression include mRNA differential display (DD), serial analysis of gene expression, subtractive hybridization, proteomics, and cDNA microarray. Using DD, we identified several upregulated genes in HCC that have clinical significance with regard to tumor proliferation and metastasis 2+-binding proteins that contain 2 Helix-E and Helix-F loop-hand Ca2+-binding motifs that mediate Ca2+-dependent signal transduction. They play important roles in many intracellular and extracellular processes, including the regulation of protein phosphorylation, enzyme activation, gene transcription, the assembly of cytoskeleton components, cell proliferation and differentiation 2+-dependent mechanism The S100 proteins are small, dimeric members of the EF-hand superfamily of Cap53 and β-catenin, which have opposing roles in tumor progression, ETR, and prognosis p53 are associated with more advanced HCC and poor prognosis β-catenin are associated with less invasive tumors and better prognosis p53 and β-catenin mutations, and to evaluate S100P as a predictive biomarker for survival in HCC patients with high tumor stage or ETR.The S100P protein was first identified in the human placenta A total of 305 unifocal, primary HCC tumors surgically resected from patients at National Taiwan University Hospital from 1982 to 1998 were used in this study. All resected tumors underwent detailed pathological assessment, and all patients received regular follow-up examinations, as described previously Surgically resected specimens were formalin fixed and paraffin embedded. Histological sections were cut at 5-µm thickness and stained with hematoxylin-eosin. All specimens were reviewed by the same investigator (HCH) to determine tumor grade and stage. The tumor grading was based on the criteriae proposed by Edmonson and Steiner 5'- AAGCTTTTTTTTTTTC-3'), 16 µM deoxynucleotide triphosphates (dNTPs), 16 µM DTT, 20 units of RNasin, and reverse transcription buffer in a 30-µL reaction volume for 60 min at 35°C. Polymerase chain reaction (PCR) was performed using 0.1 mL of reverse transcription reaction mixture and 1 unit DNA polymerase in a solution containing 2.5 µM H-T11C, 0.5 µM random primer HAP6 (5'-AAGCTTGCACCAT-3'), 1× PCR buffer, 8 mM dNTPs, and 10 µCi [-S35]dATP. The PCR procedure was performed using 40 cycles of 94°C for 30 s, 37°C for 1 min, and 72°C for 30 s, followed by a final elongation at 72°C for 5 min. After adding loading buffer to the reaction, the PCR products were heated at 70°C for 10 min, and separated by electrophoresis on a 6% polyacrylamide gel. Kodak XAR-5 film was exposed on the gels for 48 h. Bands that indicated the differential expression of mRNA were excised from the dried gel, eluted, re-amplified, cloned, and sequenced as previously described For differential display, paired mRNA samples from tumor cells and non-cancerous liver parenchyma cells from 3 low-stage (Stage I) and 6 high-stage HCCs were examined. Total RNA (2 µg) was reverse transcribed to produce cDNA using 500 units of Moloney murine leukemia virus reverse transcriptase , the H-T11C primer (5′-CTCAAGGATCTGATGGAGAA -3′) and S100P-R (5′- CCAGGGCATCATTTGAGTCC-3′). The primers for S26 were S26-F (5′- CCGTGCCTCCAAGATGACCAAAG-3′) and S26-R (5′-GTTCGGTCCTTGCGGGCTTCAC-3′).Total RNA was isolated from tissue samples and cell lines using the Trizol reagent , according to the manufacturer’s instructions. RT-PCR was used to determine the mRNA levels of S100P in the paired HCC and non-tumorous liver samples. S26 ribosomal protein mRNA, a housekeeping gene, was used as an internal control. PCR was arrested during the exponential phase for each gene (i.e. 28 cycles for S100P and 22 for S26). PCR was performed in an automatic DNA thermal cycler  with initial heating at 94°C for 2 min followed by cycles at 94°C for 30 s, 58°C for 1 min, 72°C for 1 min and finally, 72°C for 10 min. The primers for S100P were S100P-F , the slides were incubated in a 1∶200 dilution of a goat anti-S100P polyclonal antibody  at 4°C overnight. The slides are then incubated with N-Histofine Simple Stain Mouse MAX PO (G) reagent . The peroxidase activity was visualized using a diamino-benzidine tetrahydroxychloride solution , and the sections were counterstained with hematoxylin. We replaced the primary antibody with 5% FBS as a negative control. One pathologist who was blinded to patients' outcomes calculated the percentage of positive cells based on 5 independent microscopic fields (400× magnification) for each slide. For data presentation, the proportion of the tumor cells that were positive for S100P immunostaining were categorized as diffuse S100P expression (>50%), focal or heterogeneous S100P expression (10% to 50%), or S100P expression in a small number of tumor cells (1% to 10%). In the nontumorous liver, S100P protein was detected only in very few isolated liver cells. Hence, HCC with more than 1% of tumor cells showing immunostaining for S100P was regarded as positive The S100P protein was detected in formalin-fixed, paraffin-embedded sections of HCC and liver tissues using a labeled streptavidin-biotin method after antigen retrieval, as previously described p53 tumor suppressor gene were analyzed in 187 tumors by direct sequencing of the chromosomal region spanning exon 2 to exon 11, as described previously β-catenin gene were analyzed in 214 cases by direct sequencing of exon 3 in the chromosomal DNA as described previously Mutations of the All patients had been followed up for more than 5 years or until death. Among the 305 study patients, 106 (34.6%) survived longer than 5 years, and 259 (84.9%) were eligible for the evaluation of ETR. The follow-up periods for survivors ranged from 30 to 236 months . Following surgery, all patients received laboratory examinations, including assessment of serum α-fetoprotein (AFP) level, at 1- to 6-month intervals, and ultrasonography of the liver at 3- to 12-month intervals. Computed tomography (CT) and/or magnetic resonance imaging (MRI) were used to confirm and differentiate intrahepatic recurrence and/or distal metastasis in patients with clinical signs of recurrence. Cases of CT/MRI confirmed intrahepatic tumor recurrence or distant metastasis within 12 months of tumor resection were defined as ETR events, as previously described Depending on the tumor site, the tumor size, the number of tumors, the level of liver function, and the patient's condition, tumor recurrence was treated by a second resection, percutaneous ethanol injection, transhepatic arterial chemo-embolization, radiofrequency ablation, or chemotherapy. All patients had an equal opportunity to access all therapeutic modalities supported by the Bureau of National Health Insurance, Taiwan.2 test. The survival rates after tumor resection were calculated using the Kaplan-Meier method, and the difference in the survival curves was analyzed using the log rank test. A multivariate survival analysis of all the parameters that were found to be significantly correlated in the univariate analysis was performed using a Cox proportional-hazards regression model. A two-tailed P value of less than 0.05 was considered to indicate a statistically significant relationship.The data analyses were performed using the Epi Info computer software, version 7.1.0.6 . A univariate analysis was used to examine whether the immunohistochemical markers correlated with the gene mutations and the clinical and pathological parameters using the χUsing differential display, we identified a band overexpressed in high-stage HCC. The bands were excised from the gel and confirmed to be S100P by cloning and sequencing . To provThe immunohistochemical staining was used to screen 305 HCCs to determine the frequency of S100P expression and the clinical and pathological significance of S100P expression in HCC. S100P protein was not detected or detected only in very few isolated liver cells , but wasP = 0.0162), and correlated with high serum AFP . However, it did not correlate with other clinical parameters, such as age, serum HBsAg, Anti-HCV status, or tumor size. S100P expression closely correlated with high tumor grade  and high tumor stage . The Kaplan–Meier survival analysis showed S100P expression was associated with lower 5-year survival in HCC patients , tumor size (P = 1.2×10−6), tumor grade (P = 0.0456), and tumor stage (P<1×10−7) were important clinical and histopathological risk factors for ETR (P = 2.3×10−6), the presence of the p53 mutation (P = 0.0032), and the absence of the β-catenin mutation (P = 0.0011) predicted ETR. Notably, S100P expression was also a significant risk factor for ETR (P = 0.0189)  .P<0.0001), high tumor stage (P = 0.0256), and S100P expression (P = 0.0044) were independent risk factors for poor survival in HCC patients.To elucidate whether S100P is an independent factor for predicting patient survival, high tumor stage, and ETR, multivariate analyses using a Cox proportional-hazards model were performed . We founp53 and β-catenin genes are commonly mutated in HCCs p53 mutation was detected in 84 of 187 cases (44.9%), and the β-catenin mutation was identified in 32 of 214 tumors (15.0%). As shown in p53 mutation (P = 0.0032), and the absence of the β-catenin mutation (P = 0.0489). To better understand the role of S100P expression in the progression of HCC, we stratified HCC patients according to S100P, p53 and β-catenin gene status. As shown in p53 mutation had the highest frequencies of high-stage tumor (stage II and III) and ETR , which were approximately 2-fold higher than the rates of high-stage tumor and ETR in HCC cases without either S100P expression or the p53 mutation . Hence, HCC patients with S100P expression and p53 mutation had the lowest 5-year survival (P = 0.0017 (p53 mutation (+)/S100P (-) (P = 0.01) or p53 mutation (-)/S100P (+) tumors .The = 0.0017 , even wop53 mutation, the β-catenin mutation is associated with low tumor grade, low tumor stage, and better 5-year survival in HCC β-catenin mutation in HCC tumors (P = 0.0489). In addition, as shown in β-catenin-wild type HCCs with S100P expression had the highest frequencies of high tumor stage (75%) and ETR (57%), followed by HCC without S100P expression or the β-catenin mutation . HCC with the β-catenin mutation but negative for S100P expression had lowest risk of high-stage tumor and ETR . Hence, HCC patients with S100P expression and absence of β-catenin mutation had the lowest 5-year survival (β-catenin-mutated HCC or S100P (+), β-catenin-wild type HCC.In contrast to the survival , than thP = 0.0026). Similarly, HCC patients with ETR and S100P expression had a significantly lower 5-year survival rate (P = 0.0002).Because tumor stage and ETR are most important predictive factors for poor prognosis in HCC, we further analyzed the prognostic role of S100P expression in patients with high tumor stage or ETR. The combinatorial analysis showed that HCC patients with high tumor stage and S100P expression had a significantly lower -5-year survival rate  than higval rate  than HCCAlthough S100P was reported to be expressed in various types of human cancer cells Intrahepatic tumor spread through the portal vein system is the most crucial histological feature for high-stage HCC and is associated with poor prognosis We also showed that S100P protein immunoreactivity was more intense at the tumor border, and that it was more diffuse and intense in satellite nodules and portal vein tumor emboli than in the main tumor mass. Arumugam et al. reported that S100P expression correlated with cell proliferation, migration, and invasion in pancreatic cancer p53 mutation, and the absence of the β-catenin mutation were significant molecular factors for ETR. In addition, we demonstrated for the first time that HCCs with S100P expression were associated with a higher risk for ETR than HCCs that did not express S100P. The expression of S100P has also been shown to be a potential prognostic biomarker in colorectal cancer To further elucidate the impact of the expression of S100P on the incidence of ETR, we investigated possible relationships with the major clinical and pathological factors. We found that large tumor size, high tumor grade, and high tumor stage were significant histopathological predictors of ETR, and that high AFP, the presence of the p53 and β-catenin mutations p53 and β-catenin genes contribute to two distinct pathways of hepatocarcinogenesis p53 leads to aberrant mitosis, chromosome instability, more aggressive tumorigenesis, and poor prognosis in HCC β-catenin mutation is associated with low tumor grade and low tumor stage, and better patient survival p53 mutation and the absence of the β-catenin mutation. Therefore, we conducted a combinatorial analysis to further elucidate the clinical significance of the mutations with regard to S100P expression. Our findings suggest that S100P expression is associated with HCC progression, and it interacts positively with the p53 mutation, contributing to more advanced disease. This observation is similar to our previous report that stathmin expression interacts positively with the p53 mutation, and contributes to advanced HCC p53 mutation.In addition, the association of S100P with other molecular features, especially the β-catenin mutation and absence of S100P expression, which had the lowest frequency of vascular invasion (21%) and ETR (12%). Although HCC with S100P expression and the β-catenin mutation was rare in our series, occurring in only 6% of our cases, we found that the incidences of vascular invasion and ETR were higher in HCCs with S100P expression and wild type β-catenin. Further analysis showed that the majority of HCC cases with the β-catenin mutation had low tumor stage and a lower incidence of ETR, regardless of the presence or absence of S100P expression . These findings suggest that mutant β-catenin exerts a strong negative effect on tumor progression that is not abolished by S100P expression. Taken collectively, these findings indicate that S100P expression and the β-catenin mutation play opposing roles in vascular invasion and ETR in HCC. These data highlight the importance of combinatorial analysis to better understand the significance of interactions between different molecular factors in disease processes, particularly in tumor progression.We also showed that HCC with S100P expression alone had 3- and 5-fold higher frequencies of vascular invasion (75%) and ETR (57%), than those observed for HCC with the Our previous studies have shown that tumor stage and ETR are independent prognostic factors in HCC p53 mutation. These findings also highlight the potential importance of combinatorial analyses for the molecular features of HCC. The expression of S100P may identify HCC patients with high tumor stage or ETR that are at increased risk for reduced survival, aiding in the development of improved management strategies.In conclusion, our study revealed in vivo evidence that the expression of S100P is an important molecular factor for vascular invasion and intrahepatic spread, and may represent a predictive biomarker for ETR, and thus a prognosticator of unfavorable outcome. In addition, our combinatorial analysis revealed an additive unfavorable prognostic interaction of S100P expression and the


In the present study we describe sandwich design hybridization probes consisting of magnetic particles (MP) and quantum dots (QD) with target DNA, and their application in the detection of avian influenza virus (H5N1) sequences. Hybridization of 25-, 40-, and 100-mer target DNA with both probes was analyzed and quantified by flow cytometry and fluorescence microscopy on the scale of single particles. The following steps were used in the assay: (i) target selection by MP probes and (ii) target detection by QD probes. Hybridization efficiency between MP conjugated probes and target DNA hybrids was controlled by a fluorescent dye specific for nucleic acids. Fluorescence was detected by flow cytometry to distinguish differences in oligo sequences as short as 25-mer capturing in target DNA and by gel-electrophoresis in the case of QD probes. This report shows that effective manipulation and control of micro- and nanoparticles in hybridization assays is possible. In particular, many DNA-probe assays utilize oligo-conjugated magnetic particles (MPs) for capturing complementary nucleic acids ,2. TheseOur interest in the unique optical properties of QDs has resulted in their combination with microparticles for improved target capturing (solid state separation) and detection with a fast and sensitive diagnostics tool as a final goal.The synergy of combined MP-based hybridization for target capturing and QD detection techniques will extend the basic application from an analytical device to the detection of complex biological samples or to integrated lab-on-a-chip platforms. This would allow use for fast target DNA detection in point-of-care diagnostics and field analysis. However, interactions between DNA, QD probes, and MP probes are still not well defined and a detailed characterization of the procedure does not exist. A better understanding and optimization of the complex hybridization conditions between functionalized MP/QD probes and nucleic acid targets is necessary for proper assay design, and will provide new insights and possibilities in various biomedical and biotechnological fields.In this paper, we describe the design of MP-conjugated and QD-based hybridization probes and their application for the detection of the avian influenza virus (H5N1). The hybridization complex comprises three elements in a sandwich manner: (i) MPs with oligo probes on their surface for target capturing, (ii) target DNA derived from a viral sequence, and (iii) QD probes for fluorescence detection. We also addressed the question of whether hybridization efficiency is affected by oligo density on the surface of the MPs and by spacer length. The quality of probe preparation and hybridization reaction was determined by flow cytometry and fluorescence microscopy. To quantify the hybridization efficiency between the target DNA and the labeled probe, the particles-coupled DNA was directly stained with a fluorescent dye specific for dsDNA. For the discrimination of the target strand and non-complementary strand, the labeled MP–target–QD-complexes were analyzed by flow cytometry and microscopy.2.605 streptavidin conjugate.In order to investigate the usage of MPs and QDs as probes in nucleic acid hybridization assays, we designed a MP and QD-based probe assay for the detection of avian influenza H5N1. 2.1.et al. [et al. [The oligo density on the surface of MPs might influence the hybridization efficiency; on one hand a very low number of oligos reduces the number of potential binding sites, and on the other a very high density might lead to sterical hindrance. The hybridization efficiency was determined using a calibration curve of known oligo concentration. At the same time, the oligo binding capacity of MPs with spacers of different lengths was characterized to check for their potential impact on the oligo coupling . When a et al.  and Pete [et al.  reported7, 5.0 × 107, and 4.1 × 107 molecules per MP, respectively, as indicated by indirect measurement of supernatant fluorescence after conjugation. A possible effect of spacer length on target DNA and MP probes hybridization was also investigated. Based on these results  in SSC buffer. Several methods such as radioisotope, fluorescence dye labeling, or staining have been developed to reveal the hybridization efficiency [To verify the hybridization of MP probes and DNA, 1 μL of 100-mer target DNA ranging from 0.05 μM to 25 μM were hybridized on MP probes .605 streptavidin conjugates, which are surrounded by a negatively charged polymer shell, do not migrate very fast despite their charge (lane 3). Binding of biotinylated oligos (lane 2) to QD605 streptavidin conjugates increases the mobility of the particles (lane 4) due to the additional negative charge introduced by the phosphate group on the backbone of DNA. Hybridization of longer target DNA with a higher contribution of negative charges in comparison to the oligos further increases the mobility shift (lane 6). The narrow and clear band indicates the homogenity of the bound population of the target DNA. In contrast, hybridization of the non-complementary target strand (lane 7) and biotinylated oligos/QD605 streptavidin conjugate does not reveal a sharp and narrow band, which suggests a heterogeneous population of non-stringent binding (lane 8). From these results, QD probes were shown to be able to hybridize with target DNA by gel electrophoresis.Gel electrophoresis was performed to verify the hybridization of QD probes and DNA . Naked Q2.4.To investigate hybrid complex formation between functionalized MP/QD probes and different nucleic acid target lengths, the hybridization of different length DNA targets  was compared. Since the thermal stability of the QD is not well-defined ,17, onlyThe hybridization efficiency is affected by target length. Longer DNA might affect the biotinylated oligos and QD streptavidin conjugate’s access to the target. With the increasing need for knowledge about interactions of nanoparticles, microparticle-based probes and DNA hybridization, the methods to perform hybridization reactions faster and more accurately are ongoing. Furthermore, optimization of hybridization parameters for QD-probe related DNA hybridization using a microfluidic system is under development. When the target genes are captured on the MP’s surface, genes such as avian influenza virus at low concentrations can be detected. This work shows that it is possible to effectively manipulate and control micro- and nanoparticles in hybridization assays.3.3.1.8 mL−1, UMC 4N, Bangs Laboratories Inc., USA) were conjugated with amino-functionalized 25-mer oligos  that were tagged by spacers of different lengths ranging from 18 to 54 carbon chains. Conjugation was performed by carbodiimide  mediated chemistry following a Dynal Biotech protocol (http://www.invitrogen.com).Carboxy-functionalized MPs  and purified using the recommended protocol above. The complexes were stained with OliGreen and analyzed by flow cytometry. Oligo binding capacity of MPs was characterized by flow cytometry.The target sequences  were derived from a conserved region of H5N1 avian influenza virus and the non-complementary sequences were taken from the pBluescript SK(+) vector. After conjugation, the MPs were dissolved in 100 μL of PBS buffer . The efficiency of the oligo conjugation on the particle surface was determined indirectly by OliGreen  quantitation of the amount of oligos remaining in the solution phase after immobilization. The total number of particles was measured by flow cytometry adjusting flow rate and elapsed time. Thus, the binding efficiency could be calculated as the ratio of oligos to MPs. To determine the oligo density on the MP surface, 5 μL different amounts of oligos ranging from 0.005 pmol to 100 pmol with spacers of varying lengths were conjugated to 5 μL of MP  with shaking for 1 h at room temperature. After washing with 100 μL TT buffer , 100 μL TTE buffer , and 100 μL TT buffer, the hybridization complexes were dissolved in PBS and stained with PicoGreen  and analyzed by flow cytometry. The hybridization efficiency of MP probes and different amounts of target strand was obtained from a calibration curve which was recorded by measuring the average fluorescence intensity of individual particles at known concentrations of target DNA.The hybridization reaction was performed using a slightly modified procedure from Wang et al. . For the3.3.605–streptavidin conjugates  were used as fluorescent probes for different hybridization complexes: (i) Biotinylated 25-mer oligos with a 36 spacer were designed to be complementary to the oligos attached to the MPs. Biotinylated 15-mer oligos were designed to be adjacent to the MP probes, which are complementary to (ii) 40 and (iii) 100-mer target strands. Gel electrophoresis was used to verify DNA coupling to QD. For confirmation of QD and DNA interaction, QD streptavidin conjugates and biotinylated oligos were mixed together at the ratio of 1:10 and incubated in SSC buffer for 1 h at room temperature. After that, the target strand was added in equimolar amounts of QD streptavidin conjugates. The QD streptavidin conjugates/biotinylated oligos (QD probes) and the target strand hybrid were diluted in loading buffer and separated by gel electrophoresis (2% agarose in 0.5× tris-borate-EDTA buffer at 10 V/cm). The gel was illuminated and analyzed with an RAS 3,000 Image Analyzer .QD3.4.605 streptavidin conjugates (1 μM) and incubated at room temperature for 1 h. After reaction, particles were washed one more time with TT buffer and dissolved in PBS buffer for flow cytometry and fluorescence microscopy analysis.The MP probes (5 μL) were prepared and hybridized with the target ranging from 0.003 μM to 25 μM (2-fold or 4-fold diluted) as described above. The hybridization complexes were then consecutively washed with 100 μL TT buffer, 100 μL TTE buffer, and 100 μL TT buffer. After washing, they were resuspended in the 50 μL SSC buffer containing biotinylated oligos and incubated with shaking at room temperature for 1 h. The molar ratio of biotinylated oligos and target strand was 1:1. After hybridization, particles were washed twice with TT buffer, resuspended in TTL buffer containing 1 μL of QD3.5.605). Forward and side scattering were used for the separation of single particles based on size discrimination. The mean fluorescence intensity of the particles was plotted as a function of amount of the target DNA. Typically, 10,000 events per MP were recorded to analyze the data by the BD FACSDiVa software .Fluorescence images of magnetic particles hybridized with different length DNA targets were acquired using a conventional widefield microscope  equipped with a CCD camera . Ten fluorescent particles each were selected randomly for the acquisition; their fluorescence intensity was measured using the ImageJ software  and plotted as a function of the amount of DNA. At the same time, the fluorescence of the complexes was also analyzed by flow cytometry  using excitation at 485 nm and emission detection at 530 nm (OliGreen and PicoGreen) or 585 nm (QD4.In this report, the design and performance of the MP and QD probe-based method for identifying and quantifying avian influenza virus sequences was described, and detection methods for the hybridization between micro- and nanospheres with target DNA were established. We determined the oligo density on the surface of MPs and investigated spacer effects on the hybridization between MP probes and target DNA. The accessibility and affinity of the oligos to the target DNA were detected by flow cytometry. We emphasize that sequence differences as short as 25-mer in hybridization can be detected by flow cytometry. The hybridization patterns of single particles were visualized in a sandwich hybridization assay, and hybridization efficiency as a function of target strand length was presented, showing that steric hindrance prevented QD probes accessing the target strand. The established system will be used to miniaturized analytical devices for fast detection of target DNA based on MP and QD probes.


Cassava mosaic disease (CMD) is a major constraint on cassava cultivation in Africa. The disease is endemic and is caused by seven distinct cassava mosaic geminiviruses (CMGs), some of them including several variants.African cassava mosaic virus (ACMV) but share less than 89% nucleotide identity (taxonomic threshold) with any previously described begomovirus. A DNA-B genomic component, sharing 93% nucleotide identity with DNA-B of ACMV, was also characterized. Since all genomic components have a typical genome organization of Old World bipartite begomoviruses, this new species was provisionally named African cassava mosaic Burkina Faso virus (ACMBFV). Recombination analysis of the new virus demonstrated an interspecies recombinant origin, with major parents related to West African isolates of ACMV, and minor parents related to Tomato leaf curl Cameroon virus and Cotton leaf curl Gezira virus.From cassava leaf samples presenting CMD symptoms collected in Burkina Faso, four DNA-A begomovirus components were cloned and sequenced, showing 99.9% nucleotide identity among them. These isolates are most closely related to This is the first report of an ACMV-like recombinant begomovirus arisen by interspecific recombination between bipartite and monopartite African begomoviruses. Manihot esculenta Crantz), the primary food crop in sub-Saharan Africa, is severely affected by cassava mosaic disease (CMD) with losses estimated to up to 19 millions tons (1.9-2.7 billions dollars) annually [Geminiviridae: Genus Begomovirus) which are transmitted by the whitefly Bemisia tabaci (Genn.) [Cassava (African cassava mosaic virus (ACMV), East African cassava mosaic virus (EACMV), East African cassava mosaic Cameroon virus (EACMCV), East African cassava mosaic Kenya virus (EACMKV), East African cassava mosaic Malawi virus (EACMMV), East African cassava mosaic Zanzibar virus (EACMZV) and South African cassava mosaic virus (SACMV) [Sri Lanka cassava mosaic virus (SLCMV) and Indian cassava mosaic virus (ICMV), the genomes of ACMV isolates present little genetic variation and do not display evidence of recombination, suggesting that ACMV has evolved as a distinct lineage (reviewed by [CMD is endemic in Africa and seven distinct species are found associated with the disease:  (SACMV) . Several (SACMV) , many of (SACMV) . Indeed, (SACMV) -7. In coiewed by ).® Plant Minikit (Qiagen) and tested for the presence of begomoviruses using polymerase chain reaction (PCR) with degenerate primers representative of either DNA-A [In West Africa, cassava is frequently cultivated in association with several other crops such as vegetables , tubers , and cereals. In August/September 2008, leaves from cassava plants presenting CMD symptoms were collected from such a field around Ouagadougou . Total DNA was extracted using DNeasyBamHI, PstI, ApaI or NcoI and the DNA fragments of the expected size (~2.8 kb) were cloned into pGEM® vectors (Promega Biotech). Cloned genome components were sequenced by Macrogen Inc. (South Korea) by primer walking and genomic components were assembled using the DNAMAN software . Four full-length DNA-A nucleotide sequences, one from sample BF127 (BF:Oua:BF127:08) and three from sample BF128 , as well as one full-length DNA-B nucleotide sequence from sample BF127 (BF:BF127:08), were obtained (Table Ten samples showing mosaic symptoms were found to be positive for the presence of begomovirus DNA using PCR. The primary results showed the presence of ACMV and EACMV-UG in seven and one samples respectively . From twi.e. DNA-A components (2768 to 2770 nt each) with two ORFs on the viral strand (AV2/MP and AV1/CP), and four on the complementary-sense strand  and a DNA-B component (2737 nt) with one ORF on the viral-sense (BV1/MP), and one on the complementary-sense (BC1/NSP). Sequences were aligned with representative isolates of begomoviruses using the Muscle [Begomovirus (< 89% nucleotide identity for DNA-A) [The cloned genomes showed an organization typical of bipartite begomoviruses from the Old World, e Muscle  and Cluse Muscle  alignmene Muscle . Pairwisr DNA-A) , the begGGGGT) with a potential inverted repeat (GGACC), and the corresponding iteron-related domain (IRD) was identified to be MAPPRKFRIN. These features are different of those from previously described ACMV isolates (Iteron: GGAGA and IRD: MRTPRFRIQ or MRTPRFRVQ), but are similar to those found in isolates of Tomato leaf curl Cameroon virus (ToLCCMV) and Cotton leaf curl Gezira virus (CLCuGV), two monopartite begomoviruses [The putative Rep protein binding site of the four ACMBFV isolates included a single core binding site  on September 2011 and aligned as described above with our sequences. Detection of potential recombinant sequences, identification of likely parental sequences, and localization of possible recombination breakpoints was carried out using RDP [P-value cut-off of 0.05 where used. Only events detected with 3 methods or more were accepted.As these results suggested that the ACMBFV isolates could have a recombinant origin, a recombination analysis was performed on a set of 234 sequences, with 222 sequences representing the whole Old World begomovirus diversity, ten sing RDP , GENECONsing RDP , BOOTSCAsing RDP , CHIMAERsing RDP . The anaThree distinct recombination events . Consistently, phylogenetic analyses performed with the non-recombinant portion of the genome  placed ACMBFV in the cluster of ACMV  phylogenetic trees (with 100 bootstrap replicates) were constructed from full DNA components as well as alignments of both parts of the recombination event (a) using PHYML v3.0.  and the V Figure . With thV Figure . Taken tV Figure . In ordeIn conclusion, we describe for the first time an ACMV-like recombinant begomovirus arisen by interspecific recombination between bipartite and monopartite African begomoviruses. Although current data suggest that ACMV has evolved as a distinct lineage, our results prove that the ACMV genotype is also prone to recombination. Our study confirms that West Africa is likely a centre of begomovirus diversity , where rThe authors declare that they have no competing interests.FT, VSET and OT collected samples. FT, MH, MAH, ADB and JV cloned and sequenced the viruses; FT, PL and JML analysed the data and prepared the manuscript. JML, OT, NB, GK, AST, VSET and BR secured funding for the project's execution, and provided ideas and comments during manuscript preparation. All authors read and approved the final version of the manuscript.Table S1. Description of begomovirus acronyms used in the phylogenetic trees.Click here for file


One of the extraordinary characteristics of CNTs as Magnetic Resonance Imaging (MRI) contrasting agent is the extremely large proton relaxivities when loaded with gadolinium ion  CNTs. The amount of loaded gadolinium ion into the MWCNTs was quantified by inductively coupled plasma (ICP) method. To improve water solubility and biocompatibility of the system, the complexes were functionalized using diamine-terminated oligomeric poly (ethylene glycol) via a thermal reaction method.In this study equated Gdn3+ loaded PEGylated oxidized CNTs (Gdn3+@CNTs-PEG) is freely soluble in water and stable in phosphate buffer saline having particle size of about 200 nm. Transmission electron microscopy (TEM) images clearly showed formation of PEGylated CNTs. MRI analysis showed that the prepared solution represents 10% more signal intensity even in half concentration of Gd3+ in comparison with commerciality available contrasting agent Magnevist®. In addition hydrophilic layer of PEG at the surface of CNTs could prepare stealth nanoparticles to escape RES.Gdn3+@CNTs-PEG was capable to accumulate in tumors through enhanced permeability and retention effect. Moreover this system has a potential for early detection of diseases or tumors at the initial stages.It was shown that Gd Carbon nanotubes (CNTs) have unique physicochemical properties in biomedical and biological applications; hence have attracted attentions in different fields of nanotechnology -3. Large3+) with seven unpaired electrons and large magnetic moment is a suitable agent for this purpose. Although the equated Gd3+ ion is toxic, the most contrast enhancements are based on Gd3+. Chelation or encapsulation of Gd3+, decreases the toxicity of this ion for medical applications [3+-DTPA) commercially available as Magnevist®. Due to lack of specificity and sensitivity, this product is not very effective in early detection of the disease, so it has been classified as a traditional CAs [MRI is a powerful noninvasive imaging technique based on the differences between proton relaxation rates of water . To enhaications ,9. One oonal CAs .Sitharaman et al. developed the first CNT-based contrast agent. They demonstrated that Gd@Ultra-short single-walled carbon nanotubes (gadonanotubes) drastically increase MRI efficacy compared to the traditional CAs . Howevern3+ enhance contrast effect of commercial Gd. T1/T2 measurements revealed that signal intensity of Gdn3+@CNTs-PEG was more than commercial Magnevist®.In this work multi walled CNTs were functionalized by PEGylation and loaded with GdMWCNTs  were purchased from Plasmachem . CNTs were oxidized according to the procedure reported before [d before . Briefly3.6H2O  were stirred together in 100 ml deionized water and sonicated in a bath sonicator for 60 min. The solution was left undisturbed overnight whereupon the Gd3+-loaded oxidized CNTs (Gdn3+@CNTs) flocculated from the solution. The supernatant was then decanted off. To remove any unabsorbed GdCl3, remained sediment was dispersed in 25 ml of fresh deionized water with batch sonication and again, the Gdn3+@CNTs flocculated from solution was collected by decantation. This procedure was repeated 3 times. The final product was dried by vacuum oven.100 mg of oxidized MWCNTs (O-MWCNTs) and 100 mg of GdCln3+@CNTs was mixed with 474 mg Poly (ethylene glycol) bis (3-aminopropyl) terminated  and the mixture was stirred at ≈ 120°C under nitrogen atmosphere for 6 days. Upon the addition of deionized water to the mixture, the suspension was placed in a membrane tube (molecular weight cutoff ~12000) for dialysis against fresh deionized water for 3 days to remove free PEG. Dialysis phases were also collected for the confirmation of absence of free Gd3+ ion by ICP. To removing large nanotube bundles the suspension was centrifuged three times at 13000 rpm for 15 min and the supernatant was freeze-dried.28 mg of Gdn3+@CNTs-PEG.Dynamic light scattering (DLS)  was used to determine the dynamic diameter and size distributions of GdTransmission electron microscopy (TEM) and Thermal gravimetric analyses (TGA)  was applied for characterization of preparation.3(2%) and the Gd content was determined by ICP-Optical Emission Spectrometer (Varian 720-ES).For ICP (Inductively Coupled Plasma) analysis, samples should digest with strong oxidizing agents like HNO3 or concentrated H2O2. As this harsh condition is not enough for digesting MWCNTs, in this study the nanotubes were first heated in oven at 650°C for 5 h. Fallowing cooling the sample, the solid residue was dissolved in the solution of HNOThe T1- and T2-weighted spin echo images at 1.5 Tesla (repetition time/echo time 250/16 msec and repetition time/echo time 4000/64 msec) were analyzed qualitatively. The signal intensities of vials with contrast medium in solution and contrast medium in cells with the corresponding Gd concentrations were visually compared.For quantitative data analysis, the obtained MR images were transferred as digital imaging and communication in medicine (DICOM) images to a Dicom Works version 1.3.5  ,15). ForThe imaging parameters were as follows: Standard Spin Echo, # of Echoes =1, TE=15 ms, TR=100, 200, 400, 600,1000, 2000 ms, Matrix=512*384, Slice Thickness=4 mm ,FOV=25 cm, NEX=3, Pixel Band width: 130 for T1 measurements and Standard Spin Echo, # of Echoes =4, TE=15/30/45/60 ms, TR=3000 ms, Matrix=512*384, Slice Thickness=4 mm, FOV=25 cm, NEX=3, Pixel Band width: 130 for T2 measurements.T1 and T2 maps were calculated assuming mono exponential signal decay. T1 maps were calculated from four SE images with a fixed TE of 11 ms at 1.5T and variable TR values of 100, 200, 400, 600, 1000 and 2000 ms using a nonlinear function least-square curve fitting on a pixel-by-pixel basis. The signal intensity for each pixel as a function of time was expressed as follows (Equation 1) :T2 maps were calculated accordingly from four SE images with a fixed TR of 3000 ms and TE values of 15, 30, 45, and 60 ms on the 1.5T MR scanner. The signal intensity for each pixel as a function of time was expressed as follows (Equation 2):Care was taken to analyze only data points with signal intensities significantly above the noise level.One-way analysis of variance was used for comparison of the results. P values of 0.05 or less were considered as significant.n3+. Oxidizing occurred with the mixture of sulfuric and nitric acid (3:1). This procedure removes metal catalysts impurity and creates an open end termini in the structure and also sidewall defects that are stabilized by –COOH and –OH groups [3+) on the surface or inside of the interior of a CNT [In the presence study, MWCNTs were oxidized with harsh acid condition and then loaded with GdH groups ,17-19. Tof a CNT ,20. Besiof a CNT -21.3. After sedimentation and dialysis to remove unloaded Gd3+ into the oxidized MWCNTs, Gdn3+@CNTs was functionalized with PEG. ICP analysis showed the Gdn3+ content of Gdn3+@CNTs and Gdn3+@CNTs-PEG to be 4.328% and 0.02% (w/w) respectively. The absence of free Gd3+ ion in the sample was confirmed by analysis the final dialysis medium through ICP, no detectable Gd3+ was shown.The oxidized MWCNTs were loaded by soaking and sonicating them in double distilled water containing aqueous GdCln3+@CNTs was functionalized with PEG1500N (Gdn3+@CNTs-PEG). As reported by other researches, the attachment of diamine-terminated poly(ethylene glycol) with Gdn3+@CNTs were done via thermal reaction and zwitterion interaction between terminated amines of PEG and carboxylic groups of oxidized CNTs as shown in Figure Carbon nanotubes have a rigid structure and presence in bundles, so they are essentially insoluble in any solvents. As a result, solubilization of CNTs via chemical functionalization has been attracted much recent attentions ,10,17,18n3+@CNTs-PEG was more stable than Gdn3+@CNTs in PBS. The Gdn3+@CNTs-PEG remained homogeneous over 2 months of observation time whereas in the Gdn3+@CNTs black precipitation appeared after a few days . This particle size is appropriate for IV administration of solubilized gadonanotubes as a contrasting agent.The particle size of Gd3+ ions are shown in Figure n3+@CNTs-PEG image, wrapping PEG is can be clearly found around the nanotubes and the outer layer of polymer phase is discontinuous. Additionally nanotubes are dispersed either individually or in small bundles whereas in the image of Gdn3+@CNTs, tight bundles of nanotubes can be seen.Typical transmission electron microscopy (TEM) images of the functionalized MWCNTs loaded Gdn3+@CNTs-PEG and oxidized MWCNTs are shown in Figure n3+@CNTs-PEG (about 95%), content of MWCNT in this compound is low. TEM images and ICP results also confirmed this low content of MWCNT in the Gdn3+@CNTs-PEG.Thermo gravimetric analysis (TGA) and IR spectroscopy was employed to determine either the tube is wrapped by polymer chains. Thermograms and IR spectrum of GdFor oxidized MWCNTs, a weight loss was detected at 470°C, which can be attributed to the thermally unstable functional groups, e.g. –COOH and –OH on MWCNTs, formed during oxidation. These results indicate that PEG chains have successfully wrapped onto the MWCNT surfaces.n3+@CNTs-PEG solution in almost same and half concentration of Gd3+ compare to Magnevist® showed 29% and 9% more signal intensity respectively.T1/T2 measurements were performed in vitro, using magnetic resonance imaging apparatus. The analysis investigated that GdThe results of T1/T2 relaxation time (derived from equations 1 and 2) are shown in Tables n3+@CNTs-PEG clearly caused a significant decrease in both T1 and T2 relaxation time compared with Magnevist®. As shown in Table 3+ in the Gdn3+@CNTs-PEG and Magnevist® were 190.7 msec and 405.4 msec, respectively. If we depicted the 1/T1 value at different Gd3+ concentration the r1 value will be obtained 26.6 (mMol-1.sec-1) as shown in Figure 1 value for Magnevist® was only 13.4 (mMol-1.sec-1) [Gd1.sec-1) .n3+@CNTs-PEG at different Gd3+ concentrations. The r2 value for Gdn3+@CNTs-PEG was 58.8 (mMol-1.sec-1) which was greater than Magnevist®. Data in tables and T1/T2 weighted images  was performed using a 1.5T MR scanner  and a standard circularly polarized head coil . All probes were placed in a water-containing plastic container  area, and same Gd3+ concentration was 581.1 and 750.5, respectively. Therefore the signal intensity of Gdn3+@CNTs-PEG PEG was 29% and 9% more than Magnevist®, at equal or half of Gd3+ concentration, respectively.As shown in Figure n3+ -ion) clusters, carbon nanotubes have been proven to be a good candidate. Addition of polyethylene glycol to this complex could improve the expected properties of the preparation as far as its solubility, stability and more over MRI contrasting ability of them. This could be the basis for further study to reach ideal goal which is detection of any abnormal tissues or tumors at the early stages.In order to increase proton relaxivity characteristics of gadolinium ion (Gd The authors declare that they have no competing interests regarding present work.RJ conducted the experimental work and help in drafting the manuscript, FA conceived the study supervised the work and is the corresponding author of the work, SS performed the MRI experiments and ZS helped with the interpretation of the data analysis, MA helped with synthesis part of the work, RD reviewed and edited the manuscript. All authors read and approved the final manuscript.


In addition, qPCR-MN appeared to be compliant with the Percentage Law . Compared with ELISA-MN, qPCR-MN might have inherent properties conducive to reducing intra- and inter-laboratory variability while affording suitability for automation and high-throughput uses. Finally, our qRT-PCR-based approach may be broadly applicable to the development of neutralization assays for a wide variety of viruses.A microneutralization assay using an ELISA-based endpoint assessment (ELISA-MN) is widely used to measure the serological response to influenza virus infection and vaccination. We have developed an alternative microneutralization assay for influenza virus using a quantitative reverse transcription PCR-based endpoint assessment (qPCR-MN) in order to improve upon technical limitations associated with ELISA-MN. For qPCR-MN, infected MDCK-London cells in 96-well cell-culture plates are processed with minimal steps such that resulting samples are amenable to high-throughput analysis by downstream one-step quantitative reverse transcription PCR . The growth curves of three recent vaccine strains demonstrated that the qRT-PCR signal detected at 6 hours post-infection reflected an amplification of at least 100-fold over input. Using ferret antisera, we have established the feasibility of measuring virus neutralization at 6 hours post-infection, a duration likely confined to a single virus-replication cycle. The neutralization titer for qPCR-MN was defined as the highest reciprocal serum dilution necessary to achieve a 90% inhibition of the qRT-PCR signal; this endpoint was found to be in agreement with ELISA-MN using the same critical reagents in each assay. qPCR-MN was robust with respect to assay duration (6 hours  Influenza serological studies are performed frequently in the context of clinical diagnosis for identification of human infection, global surveillance for circulating and emerging virus strains, virus antigenic characterization for seasonal vaccine-strain selection, and immunogenicity evaluation for vaccine licensure Despite the obvious technical advantages associated with quantitative PCR (qPCR) in terms of sensitivity and dynamic range, few studies have exploited this methodology to measure virus neutralization in a sero-epidemiological context We wished to extend our streamlined qPCR approach to encompass RNA viruses. RNA as a PCR target, as opposed to DNA, poses additional obstacles. Nucleases present in samples that contribute to the lability of RNA must be mitigated. Additionally, one must contend with a reverse transcription step to convert the target RNA to cDNA prior to proceeding with downstream qPCR.In the present study, we demonstrate the feasibility of using a qPCR-based assessment for measuring the neutralization of an RNA virus such as influenza virus. We have emphasized an approach that minimizes sample manipulation steps so as to facilitate throughput. We make use of a commercially available reagent that allows the straightforward preparation of sample lysates from infected cells that are amenable to downstream one-step quantitative reverse transcription PCR (qRT-PCR) without the need for RNA extraction. For qRT-PCR, we routinely rely on an automated liquid handling system to prepare reactions, thereby improving throughput and data quality. The sensitivity of qPCR allows the assessment of endpoint within a single virus replication cycle. In addition, the dynamic range of qPCR allows our neutralization assay to be robust to perturbations in input virus dose. Thus, our qPCR-based assay might have inherent properties conducive to reducing intra- and inter-laboratory variability associated with existing MN assays for influenza virus while affording suitability for automation and high-throughput applications.Ethics approval by the Research Involving Human Subjects Committee (RIHSC) at the US Food and Drug Administration was obtained for the use of human sera. The sera were obtained with written informed consent from all individuals.MDCK-London cells The following viruses (propagated in embryonated chicken eggs) were obtained from V. Lugovtsev and J. Weir : IVR-148, which was derived from A/Brisbane/59/2007 (H1N1); IVR-145, which was derived from A/Solomon Islands/3/2006 (H1N1); and NYMC X-175C, which was derived from A/Uruguay/716/2007 (H3N2). These are reassortant viruses used in recent seasonal vaccine formulations. Working virus stocks for our experiments, generated after a single passage in MDCK-London cells, were aliquoted and stored frozen at −80°C prior to use.Corresponding ferret sera  were also obtained from V. Lugovtsev and J. Weir. The hemagglutination-inhibition titer was 1024 against the homologous virus for each of the three sera .et al. Adult human sera, described in Wang A microneutralization assay for influenza virus with an ELISA-based endpoint assessment was performed as described by a protocol available from the World Health Organization (WHO) 50 for each stock was calculated by the method of Reed-Muench 50, as calculated by ELISA-based titration, in 50 µL) was mixed with a dilution of serum to be tested  in a well of a 96-well culture plate. Following an incubation for 1 h at 37°C, a suspension of MDCK-London cells  was added per well. At 22 h post-infection, cells were fixed with cold 80% acetone in PBS, and virus growth in each well was assessed by ELISA as described in the WHO protocol. The neutralization titer was determined as the reciprocal of the highest serum dilution (prior to the addition of virus or cells) resulting in at least 50% inhibition of the ELISA signal.Virus stocks were titrated with MDCK-London cells as described in the WHO protocol in the presence or absence of TPCK-trypsin (1 µg/mL) using ELISA to identify culture wells positive for virus growth; the TCID50, as calculated by CPE-based titration, in 50 µL) was mixed with a dilution of serum to be tested (50 µL) in a well of a 96-well culture plate. Following an incubation for 1 h at 37°C, a suspension of MDCK-London cells  was added per well. At 6 h post-infection, virus growth in each well was quantified by qRT-PCR.The protocol for qPCR-MN was modeled after the WHO protocol for ELISA-MN. Virus stocks were titrated with MDCK-London cells at 72 h post-infection by microscopic observation of CPE. Virus inoculum (1000 TCIDLysates from MDCK-London cells grown in 96-well culture plates (in the presence or absence of influenza virus infection) were prepared using the iScript Sample Preparation Reagent  according to instructions provided by the supplier. Cells were washed once with 200 µL PBS, and 100 µL of SPR were added per well. After incubation for 2 min at room temperature (∼22°C), the resulting cell lysates were collected and stored frozen at −20°C until assessment by qRT-PCR. Remaining samples were re-frozen and stored at −20°C.AAGACCAATCCTGTCACCTCTGA; reverse: CAAAGCGTCTACGCTGCAGTCC) amplify a 104 bp product in a highly conserved region of the M1 gene of influenza A viruses The primers for qRT-PCR  were modulated while leaving others unaltered.10 range. A dilution series of the RNA standard was assessed in each qRT-PCR run in order to facilitate the quantification of influenza virus RNA copies in experimental samples.We have made use of a commercially available reagent, the Bio-Rad iScript Sample Preparation Reagent (referred to hereafter as SPR), to prepare cell lysates amenable for direct assessment by downstream quantitative reverse transcription PCR (qRT-PCR) with minimal attendant processing steps. Lysates from MDCK-London cells grown in 96-well culture plates (in the presence or absence of influenza virus infection) were prepared by washing the cells once with PBS and adding 100 µL of SPR per well. After incubation for 2 min at room temperature, the resulting cell lysates were collected and stored frozen at −20°C until assessment by qRT-PCR. In order to generate a quantification standard for qRT-PCR, total RNA was purified from MDCK-London cells infected with the influenza virus strain A/PR/8/34 and a dilution series was prepared using SPR lysate from uninfected MDCK-London cells as the diluent . The performance of our one-step SYBR Green qRT-PCR, using primers targeting a highly conserved region of the matrix gene of influenza A viruses 50 of virus, either A/PR/8/34 or a vaccine reassortant derived from A/Brisbane/59/2007 (Bris/07), was mixed with 30,000 trypsinized MDCK-London cells in suspension in the absence or presence of 1 µg/mL TPCK-trypsin. At 3, 6, 12, and 22 hours post-infection, cell lysates were prepared using SPR and subjected to qRT-PCR. The results are shown in t) at this time was 23 to 25, within the quantifiable range for qRT-PCR by a comfortable margin. By comparing the qRT-PCR signal at 3 hours and 6 hours, one can estimate that the input was amplified by >100-fold . The data support the feasibility of measuring the endpoint in a virus neutralization assay at 6 hours post-infection.Our qRT-PCR approach was used to assess the replication kinetics of influenza virus. Per well of a 96-well plate, an inoculum containing 1000 TCID50. Infection proceeded in the presence of 1 µg/mL TPCK-trypsin. At 6 hours post-infection, cell lysates were prepared using SPR and subjected to qRT-PCR. The results are shown in 50. Reasonable linearity was observed for each virus strain. Thus, our qRT-PCR approach appears to be capable of discerning 2-fold differences in virus input over a wide range. The data support the feasibility of using qRT-PCR for endpoint assessment in the context of a virus neutralization assay in which the effective virus input is modulated by the presence of neutralizing antibodies.We assessed the capability of our approach to discriminate 2-fold variations in virus input. Trypsinized MDCK-London cells  were mixed with a dilution from a 2-fold dilution series of virus  ranging from 250 to 32,000 TCID50 of virus was mixed with a dilution from a 2-fold dilution series of antiserum in a well of a 96-well plate. After allowing the neutralization reaction to proceed for 1 hour at 37°C, trypsinized MDCK-London cells  were added. TPCK-trypsin was present at 1 µg/mL. After 6 hours, cell lysates were prepared using SPR and subjected to qRT-PCR. A representative neutralization result using Bris/07 and its corresponding ferret antiserum is shown in Virus neutralization was assessed by our qRT-PCR approach using ferret post-infection sera. An inoculum containing 1000 TCIDEach of the three replicates associated with the experiment shown in qPCR-MN results (using three virus strains and their corresponding post-infection ferret sera) from four independent experiments, with three replicates for each experiment, are shown in We wished to compare our qPCR-MN results with those obtained using the established ELISA-based microneutralization assay (ELISA-MN). Per protocol 50>TCID50>TCID50.TCID50 was the input virus dose for ELISA-MN, using values determined in the presence or absence of exogenous trypsin, as appropriate, according to the experimental condition used in ELISA-MN. It is important to note that compared with our standard virus input of 1000 TCID50(CPE) for qPCR-MN, the ELISA-MN virus input of 100 TCID50(ELISA) can be greater in absolute terms depending on the experimental condition used.Per protocol, 100 TCIDELISA-MN was performed in the absence or presence of TPCK-trypsin. The endpoint was assessed at 22 hours post-infection. ELISA-MN results from three independent experiments (with each experiment consisting of three replicates) are shown in 50 virus input) were maintained as before except that neutralization was assessed in the presence or absence of TPCK-trypsin at 6, 12, and 22 hours post-infection. qPCR-MN results from three independent experiments, with three replicates for each experiment, are shown in An assessment of qPCR-MN robustness was performed with respect to assay duration. The parameters of qPCR-MN  for one of the two experiments with SI/06.An assessment of qPCR-MN robustness was also performed with respect to input virus dose. Neutralization assays were allowed to proceed for 6 hours in the presence of TPCK-trypsin; the input virus dose was 500, 1000, 3000, or 12,000 TCIDet al. Adult human serum samples  was modeled after the established ELISA-based microneutralization assay (ELISA-MN) 10; 50) The data from our study highlight several inherent advantages associated with a qRT-PCR-based approach. A concept known as the Percentage Law predicts that the neutralization titer of an antibody solution would not be impacted by the input virus dose, assuming that the antibody is in vast molar excess relative to the virus during a neutralization reaction  bona fide single-cycle assays such as MN assays using pseudotype reporter viruses vs. 12 hours , are associated with greater variability compared with ELISA-MN of 18–22 hour duration The sensitivity of qRT-PCR allows endpoint assessment for qPCR-MN at 6 hours post-infection compared with ∼18–22 hours for ELISA-MN. A shorter duration for an assay is desirable in itself. However, aside from logistics, there may be a theoretical basis to prefer an endpoint assessment at 6 hours. Currently available neutralization assays for influenza virus, including ELISA-MN, are associated with a significant degree of intra- and inter-laboratory variability 12 hours , 5, 6. A12 hours , 5, 6. T12 hours . OverallMost laboratories now appear to perform ELISA-MN in the absence of exogenous trypsin Endpoint measurement by qRT-PCR might be more amenable to standardization across laboratories compared with ELISA. Reference reagents (purified RNA or SPR-derived cellular lysates) can be stored frozen and distributed in order to facilitate the establishment and optimization of qRT-PCR in a new laboratory. In contrast, a comparable reference antigen (influenza virus nucleoprotein) for ELISA would be less straightforward to generate.The availability of the option to freeze qPCR-MN samples would also be useful in other circumstances. In case of a technical failure that occurs during ELISA for ELISA-MN, one would have to start again from the beginning with virus neutralization and cell infection. A comparable failure during qRT-PCR for qPCR-MN would simply necessitate a re-assessment of frozen samples.For our qPCR-MN, preparation of sample cell lysates is accomplished manually in a few minutes. For downstream qRT-PCR, we routinely make use of an automated liquid handling system to prepare the reactions in 96-well plates. While not essential for our qPCR-based approach, automation facilitates throughput, improves data quality, and reduces cost . While each individual step in ELISA is simple in nature, full automation might be more difficult to achieve because of the higher overall number of manipulations required.It is important to note that ELISA-MN has a distinct advantage over qPCR-MN in terms of cost . Thus far, we have largely focused on technical feasibility and logistical concerns aside from cost. However, the current cost associated with qPCR-MN might be acceptable for many applications. In addition, it might be possible to achieve further cost reduction by additional downscaling. Finally, market dynamics of supply/demand might, in time, result in lower reagent costs.We conclude by commenting on the general applicability of the qRT-PCR-based approach. It may be possible to develop qRT-PCR-based neutralization assays for other RNA viruses in a relatively rapid manner. Specific reagents that are difficult or time-consuming to generate, such as antibodies or recombinant reporter constructs, are not required in an assay using qRT-PCR. As noted earlier