Dear Dechassa Tegegne!

Here is my answer to your question. Please check it and came back to me for more explanations.

Sincerely yours!

Dr. Bratko Filipič

Email: [email protected]

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Materials and Methods

Specimens.

Samples were randomly selected among positive nested PCR serum/plasma specimens from service testing performed on patients admitted to Queen Sirikit National Institute of Child Health (QSNICH) between 2000 and 2002. Acute specimens were collected from patients with a history of fever and meeting at least one of the following additional criteria: positive tourniquet test, leukopenia, or bleeding manifestation.11Each sample was aliquoted when delivered to the laboratory and stored at −70°C and previously unthawed were used for PCR and viral isolation. All patients were serologically confirmed as acute primary or secondary dengue infections. Of these samples there were 644 DENV-1, 499 DENV-2, 302 DENV-3, and 79 DENV-4 (1,544 total), representing 488 DF and 959 DHF according to World Health Organization (WHO) established criteria.

Detection of virus genome by reverse transcriptase (RT)-PCR.

The RT-PCR was performed according to the protocol of Lanciotti and others12 with modifications as described by Klungthong and others.13

Virus isolation in C6/36 cells and identification of serotypes.

The PCR-positive serum specimens were used to infect C6/36 cell cultures.14–16 Original serum or plasma (0.3 mL) was blindly passaged three times on C6/36 cell culture with a 7-day incubation period for each passage. Following the third passage, the culture fluid was tested against a panel of monoclonal antibodies against each of the four dengue virus serotypes.16

Mosquito amplification.

All samples that could not be recovered by C6/36 cell culture were intrathoracically inoculated with 0.34 μL of the clinical sample into 15–20 live T. splendens mosquitoes.17–20 After 14 days, ∼10–15 surviving mosquitoes were tested by head squash and immunofluorescent antibody assay (IFA) for flavivirus antigen. Bodies of virus-positive mosquitoes were triturated and passaged once in C6/36 cell culture as described previously. The virus present in culture fluid was then serotyped as above.

Dengue/Japanese encephalitis (JE) IgM/IgG enzyme immunoassay (EIA).

All serum/plasma were tested for dengue and JE IgM and IgG by Armed Forces Research Institute of Medical Sciences (AFRIMS) antibody capture EIA to serologically confirm the diagnosis and to differentiate primary versus secondary dengue infection.21 For specimens, 40 units (U) of anti-dengue IgM (with anti-dengue IgM greater than anti-Japanese encephalitis virus [JEV] IgM) were considered evidence of acute dengue infection. From paired sera (acute and convalescent interval of ≥ 7 days), a dengue IgM-to-IgG ratio ≥ 1.8 defined a primary dengue virus infection. A ratio < 1.8 defined a secondary dengue virus infection. With serial specimens, a 2-fold increase in IgG to dengue with an absolute value of ≥ 100 U indicated a secondary infection in the absence of anti-dengue IgM of ≥ 40 U.22–24

Statistical analysis.

Data were entered and manipulated using FoxPro for Windows software (Microsoft, Redmond, WA) and analyzed using SPSS for Windows version 12.0 (SPSS Inc., Chicago, IL) and SAS analytic software, version 9.1 (SAS Institute, Inc., Cary, NC). The χ2 analysis was done for contingency tables. Logistic regression was used for multivariate analysis. All variables that were significantly associated with isolation positivity by bivariate analysis were initially entered as predictors in the multivariate regression model. The best model was selected by the method of backward elimination, in which the variable with the highest Pvalue greater than a chosen cut-off (we selected P = 0.10) is removed at each step until all remaining variables in the model have P values less than the cut-off.

Hi Dechassa,

I'm not exactly sure what you are asking here, but I think you are wondering what the differences are in a virus in an infected mosquito vs once it has been passaged in a cell line - in essence, what is the difference between in vivo vs in vitro.

The important thing to remember is that culturing a virus in vitro will alter the genetic sequence of the virus - the error-prone polymerases of viruses result in viral heterogeneity (what some people refer to as 'qausispecies') which allow for adaptation to disparate cellular environments. Although you are inferring that you will be attempting isolation in the mosquito C6/36 cell line, this is not truly representative of an in vivo system (ie a wild mosquito) for several reasons including the decificent RNAi pathway in these cells.

If you are trying to isolate virus from wild mosquitoes, I would recommend you attempt this on both mosquito and a mammalian cell line if you are looking for arboviruses as the virus may replicate better in mammalian cells (this mimics the natural replication cycle plus repeated culture in the same cell type often has fitness effects and can even attenuate the virus!). Therefore, many researchers attempt viral isolation of arboviruses in mosquitoes in mammalian cells. The down side to this is that it will introduce more sequence variation the the original virus as the virus adapts to the new cellular environment.

If you are not willing to use mammalian cells, then C6/36 may work, although you will probably need blind passing and frequent monitoring with a PCR assay to ensure sufficient replication.

Any system you use to try and isolate the virus will likely alter the sequence, therefore you cannot assume the sequence is the same as was in the original wild caught infected mosquito. Of course you can try and sequence directly from the mosquito, but there might not be a lot of starting material to sequence from!

In summary:

DIRECT SEQUENCING: True sequence identity but difficult to achieve due to low amount of sample

CULTURE IN C6/36: May produce more sample (making sequencing easier) although will probably require serial blind passaging. Unlikely to significanly alter the viral sequence

ALTERNATE CULTURE IN MAMMALIAN/MOSQUITO CELLS: Most likely to result in an isolated virus, but most likely to introduce variation

So it really depends on what you want - if it is an isolated virus or highest chance of good sequence data, try culturing in a mammalian cell line (then mosquito if needed). If you want to know the exact sequence in a wild mosquito, try and sequence directly.

Hope that helps

Thanks Dr.Barry Atkinson for very important information I want to isolate USUV and WNV from mosquitoes.