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Investigation of the role of the ubiquitin proteasome pathway in dengue virus life cycle 4

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In this study, we detail important differences in the measurement of viral growth kinetics in Vero and C6/36 tissue cultures, in Aedes aegypti mosquitoes, and in viremic human sera using

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of components of the UPP to alleviate ER stress, the inhibition of which leads to the decoupling of infectious virus production from RNA replication

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Figure 4-1 Schematic diagram of findings 1 Proteasome inhibitors prevent

polyubiquitylated misfolded proteins to be degraded by the proteasome 2-3 Accumulation of misfolded proteins stresses the cell and activates the PERK pathway 4 Activation of this pathway phosphorylates eIF2α 5 Translational attenuation occurs and results in decreased levels of TC10 and EXOC7 6 Dengue egress is inhibited due to the reduction in protein levels of these host factors necessary for virus egress

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4.2 Unfolded protein response during flavivirus infection

The ER is an extensive membranous network that serves different specialized

functions, such as intracellular signal transduction and calcium storage In addition, most secreted and transmembrane proteins enter the lumen of the ER prior to being secreted for maturation To maintain homeostasis in the ER, the ER has evolved three mechanistically distinct pathways collectively called the UPR

Under ER stress, ER chaperone immunoglobulin heavy chain binding protein (BiP), plays a central role in the activation of PERK, activating transcription factor 6 (ATF-6) and the ER transmembrane protein kinase/endoribonuclease (IRE-1) pathways (Ron & Walter, 2007; Schroder & Kaufman, 2005; Shen & Prywes, 2005) PERK is

an ER-localized type I transmembrane protein and the PERK pathway functions as a first-response mechanism induced by various stressors and attenuates global protein synthesis via phosphorylation of eIF2α (Wek et al, 2006)

PERK activation also induces the activation of C/EBP homologous protein (CHOP) and growth arrest and DNA damage-inducible protein (GADD34) In the case of sustained ER stress, CHOP is responsible for apoptosis of the cells Alternatively, CHOP can also function as a pro-survival transcription factor leading to induction of GADD34, a subunit of protein phosphatase 1c (PP1c) that targets the

dephosphorylation of phosphorylated eIF2α (eIF2α-P) (Rutkowski et al, 2006) Failure to suppress ER stress leads to activation of the ATF6 and IRE1 pathways, which act to alleviate the accumulation of misfolded proteins by up-regulating host factors that increase the capacity of the ER to handle the synthesis of nascent proteins

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(Ron & Walter, 2007)

The life cycle of DENV and other members of the Flaviviridae family depend heavily

on the host ER to translate, replicate, and package their genome (Lindenbach et al, 2007) If ER stress hampers the completion of its life cycle, DENV must thus be able

to modulate the ER stress response to survive Indeed, several studies have shown that DENV infection modulates the ER stress response in a time-dependent manner

(Paradkar et al, 2011; Pena & Harris, 2011; Umareddy et al, 2007) (Yu et al, 2006) Early DENV2 infection has been shown to trigger and then suppress PERK-mediated eIF2α phosphorylation, and the IRE1 and ATF6 pathways were then activated in the later stages of infection (Pena & Harris, 2011) Consequently, inhibition of the

proteasome could induce additional UPR that then overcomes the ability of DENV to regulate ER stress Critically, inducing ER stress with an agonist without inhibiting proteasome function recapitulated the observed down-regulation of EXOC7 and TC10 protein levels along with the decoupling of infectious virus production from viral RNA replication Supporting our data, salubrinal, a drug that inhibits eIF2α

dephosphorylation, thereby increasing phosphorylated eIF2α levels was previously shown to reduce the production of infectious viruses (Umareddy et al, 2007)

The activation of individual branches and components of the UPR by other members

of the Flaviviridae family have also been reported Studies with hepatitis C virus have

demonstrated activation and suppression of the PERK pathway in a time-dependent manner (Pavio et al, 2003) Also, infection with Japanese encephalitis virus was shown to induce the IRE pathway, protecting cells from virus-induced cytopathic effects (Yu et al, 2006), whereas WNV induction of the UPR was shown to lead to

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CHOP-mediated apoptosis (Medigeshi et al, 2007) Taken together, these studies suggest that flaviviruses have evolved to manipulate the UPR, rendering the UPR as a potential anti-viral host target However, no therapeutic that specifically targets the three sensors of the unfolded protein response, has been licensed, likely due to the cellular toxicity it may cause

4.3 Repurposing proteasome inhibitors as an anti-flaviviral therapeutic

On the other hand, proteasome inhibitors are currently indicated in the treatment of certain hematological malignancies The much greater sensitivity of myeloma cell lines and mantle cell lines to proteasome inhibition compared with normal peripheral blood mononuclear cells is poorly understood The UPP plays a critical role in

regulating ER stress to enable DENV to complete its life cycle by egressing cells

through exocytosis Perturbing this pathway is utilized by the Ae aegypti midgut to

inhibit continued infectious DENV production without harm to itself and this same approach could potentially be exploited as a therapeutic strategy in dengue

Indeed, the potency of proteasome inhibition as an anti-dengue strategy is suggested

by the low nanomolar EC50 of bortezomib, one of the first FDA-approved proteasome inhibitor, in DENV-infected primary monocytes Similarly, bortezomib treatment in

an immunocompetent mouse model was able to reduce plasma leakage, the degree of thrombocytopenia as well as the pro-inflammatory responses At a mechanistic level,

we have demonstrated that proteasome inhibition could inhibit virus egress and hence spread within mammals With a good understanding of its antiviral action, the

potential of bortezomib or other proteasome inhibitors to serve as an anti-dengue or

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even anti-flaviviral therapy will need to be explored in clinical safety and efficacy trials Indeed, a known side effect of bortezomib is thrombocytopenia although this is only observed in multiple myeloma patients after weeks of continuous treatment As treatment for dengue would not exceed a week, the side effects observed only after prolonged therapy may not be clinically relevant for dengue Therefore, it is also unlikely that bortezomib would exacerbate the situation in patients who present with the symptoms of thrombocytopenia.

given subcutaneously or intravenously to patients This is not ideal for any dengue therapeutics, as injections are not recommended for dengue patients having the tendency to bleed Opportunely, this problem can be circumvented by the recent introduction of ixazomib, the first oral proteasome inhibitor that is currently

anti-undergoing Phase 3 clinical studies (Moreau, 2014) Table 4-1 lists the current

development of various proteasome inhibitors undergoing different phases of clinical trials

While we have demonstrated the inhibition of virus egress as the antiviral mechanism effected by proteasome inhibition, it is interesting that proteasome inhibitors may have other modes of antiviral action The UPP has also been shown to be critical for the life cycle of Nipah virus Inhibition of the proteasome led to impaired nuclear export of the viral matrix protein to the cytoplasm (Wang et al, 2010) Studies on retroviruses have also demonstrated that disruption of the proteasome function

depletes the free ubiquitin pool (Mimnaugh et al, 1997), which is necessary for the ubiquitylation of late domain on Gag protein for proper viral budding (Patnaik et al, 2000; Schubert et al, 2000) While such mechanisms could contribute to our observed inhibition of DENV replication, our findings suggest another complementary

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mechanism that impairs flaviviral life cycle Altogether, proteasome inhibition could

be a broad spectrum antiviral approach against viruses that egress cells via exocytosis

or which requires ubiquitylation to regulate the functions of specific viral proteins

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Table 4-1 Current developments of proteasome inhibitors undergoing different phases of clinical trials

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4.4 Learning from Ae aegypti mosquito

Unlike infection in humans, dengue infection in mosquitoes is nonpathogenic It is interesting that the mosquito midgut down-regulates several components of the UPP naturally to inhibit persistent infectious DENV production One explanation may be transcriptomic changes in the mosquito midgut upon ingestion of a bloodmeal or during its gonotrophic cycle Both UBE2A and DDB1 act upstream of the proteasome

in the UPP; the former belonging to the E2 ubiquitin-conjugating enzyme family (Jentsch et al, 1987), and the latter functioning as an adaptor molecule for the cullin 4-ubiquitin E3 ligase complex (Higa et al, 2006) UBE2A targets several short-lived regulatory proteins for polyubiquitylation and subsequent turnover by the 26S

proteasome (Jentsch et al, 1987) DDB1 has been shown to facilitate the

ubiquitination and subsequent proteasome-mediated degradation of STATs for the

Rubulavirus genus of Paramyxoviridae (Precious et al, 2005; Ulane et al, 2005)

Our findings are concordant with previous studies in female mosquitoes where

various UPP-specific genes such as TSG101 (AAEL012515), NEDD4 (AAEL002536) and SCF ubiquitin ligase (AAEL004691) have also been identified as critical host

factors for DENV replication (Guo et al, 2010; Mairiang et al, 2013; Sim &

Dimopoulos, 2010) Due to genetic variability across various mosquito strains and DENV-2 strains, as well as variation in the methodology of the experiments such as data analysis and time-points used, it is unsurprising that the UPP-specific genes detected in these studies were not the same individual genes As these molecules function to signal for the activation of the effector of the UPP, the proteasome, we reasoned that DENV is dependent on the pathway rather than signaling intermediates

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for successful completion of its life cycle Down-regulation of several genes in the UPP, along with the midgut barrier (Gomez-Machorro et al, 2004), forms a major part

of the mosquito’s response that restricts DENV infection Multiple quantitative trait loci have been associated with the midgut barrier, but the operating mechanisms of specific genes involved remain to be fully determined (Black et al, 2002) Mosquito genes and physiological pathways related to innate immunity, redox activity, fat, protein and carbohydrate production and metabolism were found to be modulated in response to DENV infection (Behura et al, 2011; Tchankouo-Nguetcheu et al, 2010) These observations come from multiple studies of specific mosquito tissues and time

points following infection, and used different combinations of Ae aegypti strains and

DENV-2 strains It will be interesting to analyze the RNAseq data on the mosquito midgut transcriptome during DENV infection and compare these results with existing data available Studying how these responses limit DENV infection without any apparent harm to the mosquito (Fragkoudis et al, 2009), which contrast with the human host response to DENV that is intimately linked with dengue pathogenesis (Whitehorn & Simmons, 2011), offers a hitherto unexplored opportunity for

therapeutic discovery

4.5 Beyond the anti-viral effects of bortezomib: Potential use as an adjuvant

In the scope of this thesis, we show that bortezomib inhibited virus egress and

induced apoptosis in primary monocytes This raises the possibility that DENV

‘trapped’ in the cells could firstly, increase MHC presentation of viral antigens in infected cells and secondly, allow cross-presentation of viral antigen via apoptotic cells The former is unlikely because bortezomib was demonstrated to down-regulate

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cell surface HLA (human leukocyte antigen) class I expression of monocytes (Shi et

al, 2008) Preliminary results from our studies suggest that although inflammatory responses were reduced in bortezomib-treated mice, IFNγ levels were increased instead This is suggestive of elevated T cell and NK (natural killer) cell activity and future work could be performed to examine if bortezomib could indeed induce NK cells, T cells and antibody responses against DENV in immunocompetent mice If this possibility is true, it is plausible to use bortezomib as an adjuvant to raise adaptive immunity when co-injected with partially attenuated DENV The antiviral action of bortezomib will limit the number of cycles of infection in the host while enhancing the acquired immune response against DENV

4.6 Conclusion

In conclusion, the UPP plays a critical role in regulating ER stress to enable DENV to complete its life cycle by egressing cells through exocytosis Perturbing this pathway

is utilised by the Aedes aegypti midgut to inhibit continued infectious DENV

production without harm to itself and this same approach could potentially be

exploited as a therapeutic strategy in dengue

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References

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Appendix A

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Am J Trop Med Hyg., 89(5), 2013, pp 1001–1005

doi:10.4269/ajtmh.13-0100

Copyright © 2013 by The American Society of Tropical Medicine and Hygiene

Short Report: Comparison of the Mosquito Inoculation Technique and Quantitative Real Time

Polymerase Chain Reaction to Measure Dengue Virus Concentration

Milly M Choy, Brett R Ellis, Esther M Ellis, and Duane J Gubler*

Signature Research Program in Emerging Infectious Diseases, Duke-National University of Singapore Graduate Medical School,

Singapore, Republic of Singapore

Abstract An accurate measure of infectious dengue virus in human and mosquito tissues is critical to fully understand virus–host relationships, disease severity, viral fitness, and pathogenesis In recent years, RNA copy number measured

by quantitative real time-polymerase chain reaction has been used to measure dengue virus concentration in vitro and

in vivo In this study, we detail important differences in the measurement of viral growth kinetics in Vero and C6/36 tissue cultures, in Aedes aegypti mosquitoes, and in viremic human sera using RNA genomic equivalents and mosquito infectious dose 50 (MID 50 ) Although there was reasonably good correlation between the two methods, RNA copy number was 2 to

5 logs greater than infectious virus titers These differences varied significantly depending on virus strain, viral platform, infectious virus assay, and viral growth phase The results have important implications for the correct interpretation

of biological and epidemiological data from experimental and clinical studies, and show that genomic equivalents should

be interpreted with caution when used as a proxy for infectious virus in such studies.

Epidemic dengue/dengue hemorrhagic fever (DF/DHF) has

emerged as the most important mosquito-borne viral disease of

humans in the past 40 years with both the viruses and mosquito

vectors spreading globally in the tropics.1This spread has been

closely linked to the global trends of urbanization and

globali-zation, combined with a lack of effective mosquito control.

Most urban centers of the tropics are now hyperendemic with

multiple virus serotypes co-circulating The result has been

larger and more frequent epidemics associated with more

severe disease 1

From the 1940s when dengue viruses (DENV) were first

isolated 2,3 until the 1960s, scientists relied on suckling mice

for isolation and assay of DENV In the 1960s, mammalian

cell cultures were used,4but both methods were highly

insen-sitive to primary DENV isolates that had not been adapted by

serial passage.5 Subsequent development of the mosquito

inoculation technique and the C6/36 Aedes albopictus cell line

in the 1970s provided significant improvement in sensitivity,

and permitted work with unpassaged DENVs.5–7 However,

the relatively insensitive plaque assay that measures

plaque-forming units (PFU)8 has continued to be used to measure

infectious DENV in experimental studies, and the C6/36 cell

culture system has been primarily used for virus isolation6–9

because most virology laboratories lacked the ability to

work7,8,10with live mosquitoes.

The efficacy of clinical diagnosis, surveillance, prevention,

and control of dengue has been limited by the lack of easy to

use and sensitive diagnostic tests Currently, laboratory

diag-nosis in most dengue-endemic countries relies on detecting

immunoglobulin M (IgM) antibody in acute serum samples.

More recently, commercial tests combining NS1 antigen and

IgM antibody detection have become increasingly popular.11

For DENV detection and quantitation, quantitative real time

polymerase chain reaction (qRT-PCR) has become the method

of choice in the past 20 years12–15; this method is generally

more sensitive and efficient than isolation assays, and can

provide a rapid serotype-specific diagnosis Moreover, DENVs can be identified and quantified directly from clinical samples Although qRT-PCR measures RNA and not infectious virus, qRT-PCR has been increasingly used in recent years to mea- sure DENV titers.14,15

Although qRT-PCR has been compared with the relatively insensitive plaque assay (PFU),16–20the actual ratio of RNA copy number to infectious virus remains unclear Moreover,

it is not known to what degree the infected host, the virus strain, or time of infection may influence that ratio To better define the quantitative and biological relationships between RNA copy number and infectious DENV, we compared qRT-PCR with the titer of infectious DENV measured by the mosquito inoculation technique (mosquito infectious dose 50, MID 50 ), which is the most sensitive biological assay available for measuring unpassaged infectious DENVs Quantitative comparisons were performed using viremic human sera, infected mosquitoes, vertebrate, and mosquito cell cultures Two low passage DENV-2 strains (PR1940 and PR6913) with contrasting virus replication kinetics (Manokaran and others, 2013, manuscript submitted) isolated during a

1994 epidemic in Puerto Rico, were used in the mosquito and

in vitro cell culture experiments DENV-2 viremic sera were obtained from patients during a 2011 epidemic in Pakistan.21The DENV-1, -3, and -4 were isolated from patients during routine surveillance in Singapore and Indonesia.22,23 All patient samples were collected with patient consent and insti- tutional review board approval.

Aedes aegypti mosquitoes were obtained from a colony at the Duke-NUS Graduate Medical School The colony was established in 2010 with specimens collected in Ang Mo Kio, Singapore, and supplemented monthly with field-collected mosquitoes (10% of colony) to maintain genetic diversity To investigate virus kinetics, 1- to 5-day-old female Aedes aegypti mosquitoes were inoculated with 100 MID 50 of each virus5and incubated at 28 °C Infected mosquitoes were harvested at Days 3, 7, 10, 14, and 17 post-infection Surviving mosquitoes were killed by freezing and stored at −80°C until assayed Both C6/36 and Vero cell cultures were inoculated with 0.1 multiplicity of infection of each virus Cell culture supernatants were harvested on Days 1, 3 and 7 post-infection and stored at

−80°C until assayed.

*Address correspondence to Duane J Gubler, Signature Research

Program in Emerging Infectious Diseases, Duke-NUS Graduate

Medical School, 8 College Road, Singapore 169857 E-mail: duane

.gubler@duke-nus.edu.sg

1001

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Three individual mosquitoes at each time point were

tritu-rated and tittritu-rated by qRT-PCR, mosquito inoculation and

plaque assay, and the mean titer calculated Cell culture

supernatants at each time point were titrated by qRT-PCR

and mosquito inoculation only For the MID 50 assay, virus

titrations were performed by making 10-fold serial dilutions

of each of the virus suspensions and viremic sera in Leibovitz’s

L-15 medium Dilutions were inoculated intrathoracically

into six male mosquitoes and held for 10 days at 28 °C, after

which surviving mosquitoes were harvested and stored at

−80°C 5

Harvested mosquitoes were examined for the

pres-ence of viral antigen in brain tissues by indirect

immuno-fluorescence on mosquito head squashes24and the MID 50 per

mosquito or per mL was calculated by the method of Reed and

Muench.25 For plaque assay,8 serial dilutions of each virus suspension were inoculated in triplicate onto BHK-21 cells and incubated for 1 h at 37°C Media was aspirated and replaced with 0.8% methyl-cellulose in maintenance medium After 6 days at 37°C, cells were fixed with 20% formaldehyde and stained with 1% crystal violet The plates were washed and dried, and the PFU per mosquito or per mL were counted Viral RNA quantitation was performed following RNA extraction of viremic sera, infected-mosquito tissues, and cell culture supernatants using the QIAamp Viral RNA Mini kit (Qiagen, Hilden, Germany) A one-step qRT-PCR was per- formed using the SuperScript III Platinum One–Step Quanti- tative RT-PCR System (Invitrogen, Carlsbad, CA) The RNA copy number was calculated by generating a standard curve

Figure 1 Replication kinetics of DENV-2 (A) PR1940 and (B) PR6913 in adult female Aedes aegypti mosquitoes Virus titers are measured by plaque assay (PFU/mosquito ▲), mosquito inoculation technique (MID 50 /mosquito ü), and qRT-PCR (RNA copy number/mosquito…) Each point represents the mean of three mosquitoes triturated individually and the error bars indicate standard error of the mean.

Figure 2 Replication kinetics of DENV-2 PR1940 and PR6913 derived in cell cultures measured by the mosquito inoculation technique (MID50/mL ü) and quantitative real time polymerase chain reaction (qRT-PCR) (RNA copy number/mL…) ( A) PR1940 in Vero cell culture ( B) PR1940 in C6/36 cell culture (C) PR6913 in Vero cell culture (D) PR6913 in C6/36 cell culture Each point represents the mean of three biological replicates and the error bars indicate standard error of the mean.

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from a plasmid control containing the region of interest 13 ;

the primers designed for qRT-PCR target the region NS5

for DENV-1, E for DENV-2, prM for DENV-3, and E for

DENV-4.

All results represent the average of three biological

repli-cates A two-tailed unpaired Student’s t test was used to

deter-mine if the difference in the means was statistically significant.

Linear regression analysis was used to determine if MID 50

titers correlated with the RNA copy number (P < 0.05), and

calculations equivalent to analysis of covariance was used to

assess differences between slopes (GraphPad Prism v5.0,

GraphPad Software Inc., La Jolla, CA).

The replication kinetics of DENV in live Aedes aegypti

mosquitoes, Aedes albopictus mosquito cell cultures, and

Vero mammalian cell cultures showed that RNA copy

num-ber was typically 2–3 logs greater than the MID 50 titer,

regardless of the host tissue or cell culture from which the

virus was harvested (Figures 1 and 2) When titers per whole

mosquito were compared, the RNA copy number was 100 to

1,000 times higher than the MID 50 titer, which was 100 to

1,000 times higher than the PFU measured by plaque assay

(Figure 1) This difference was evident for both DENV-2

strains (PR1940 and PR6913), regardless of the maximum

titers observed in all assay platforms.

In general, linear regression showed that the RNA copy

number was correlated with MID 50 titers for DENV-2 in

mos-quitoes (P < 0.0001, R 2 = 0.567) and cell cultures (P < 0.0001,

R 2 = 0.950) (Figure 3A) However, the slopes differ

signifi-cantly (P < 0.001, F = 13.95), showing that the ratio of RNA

copy number to infectious virus may differ when using

differ-ent host systems to grow DENV Although there is a tively good general correlation between the MID 50 titers and RNA copy number using the same host systems to grow DENV, the accuracy of measuring infectious DENV using RNA copy number may vary based on the virus strain or time

rela-of infection as the ratio may be significantly different from one another (Figure 3B and C) Different conversion ratios were also shown for different serotypes of DENV, with 7 day old C6/36 virus supernatants for DENV-1, DENV-3, and DENV-4 showing 2.0, 0.7, and 2.5 logs higher concentrations

by qRT-PCR, respectively (Table 1) Of interest, DENV-3 concentrations varied by only 0.7 log between the two methods This small difference could be a unique replication characteristic of that virus strain or result from the specific time in viral growth when it was sampled Clearly, more strains of all four serotypes should be tested.

A greater variation was observed when measuring DENV-2 viremias in human sera using the two methods, varying by 2–5 logs, depending on the individual serum (Figure 4) No correlation was observed between RNA copy number and infectious virus titers for human sera (P = 0.3109).

Figure 3 (A) RNA copy number versus MID 50 in Aedes aegypti mosquitoes (…) and cell cultures ( ○) The regression equations are DENV-2 copies = 0.653 MID 50 + 4.93 (R 2 = 0.567) and DENV-2 copies = 1.05 MID 50 + 2.14 (R 2 = 0.950), respectively The two slopes are significantly different ( B) Ratio of genomic equivalents (GE) to MID 50 at different time-points for PR1940 and PR6913 in mosquitoes ( C) Ratio of genomic equivalents (GE) to MID at different time-points for PR1940 and PR6913 in cell cultures.

Table 1 Comparative titration of C6/36 cell culture virus supernatants by qRT-PCR and mosquito inoculation *

DENV serotype Copy number/mL MID 50 /mL Log difference (P value)

*qRT-PCR = quantitative real time polymerase chain reaction.

COMPARISON BETWEEN MID50 AND COPY NUMBER OF DENV 1003

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This is the first direct comparison of RNA copy number

measured by qRT-PCR, and infectious DENV titer measured

by the mosquito inoculation technique, in vitro and in vivo.

Our results agree with previous studies, which show positive

correlations between flavivirus RNA copy number and

infec-tious virus in cell cultures and Aedes aegypti mosquitoes.16–20

A consistently higher, but variable RNA copies to infectious

virus titer ratio is likely caused by the presence of

noninfec-tious immature virions or defective virus particles.16–20,26,27

However, the differences in ratio could also be caused by

intrinsic variation in virus replication or translational

efficien-cies in different host tissues Of importance was the lack of

correlation between RNA copy number and infectious virus

titers in human sera Viremia (infectious virus) in humans is

influenced by the strain of virus, the day of infection the

serum was collected from the patient, and the individual’s

previous dengue experience, which influences the innate and

adaptive immune response and thus, the production of

noninfectious defective virus particles Infectious virus titers

can also be influenced by how the serum is processed after the

blood draw, the number of freeze-thaw cycles, and the storage

temperature and how it is shipped It should be noted,

how-ever, that the sera used in these experiments were processed

immediately after the blood draw, stored at −80°C, and

shipped frozen on dry ice to Singapore; the sera were never

thawed before shipping to Singapore.

In conclusion, we show that RNA genomic equivalents are

not a reliable proxy for infectious virus as the host, the virus

strain, and time of infection may influence the ratio of

geno-mic equivalents to infectious DENV Although there was a

reasonably good correlation between the two methods in

measuring virus concentration, caution must be exercised in

generalizing about infectious virus and the interpretation of

results in both clinical and experimental studies An accurate

measure of infectious virus is critical to understanding dengue

virus biology and pathogenesis, and for development of

effec-tive diagnostic tests, vaccines, and therapeutics Thus,

although qRT-PCR is a highly sensitive and useful DENV

diagnostic tool, quantitation of infectious DENV, especially

from sera, autopsy tissues, and mosquitoes should ideally be

performed using the mosquito inoculation assay, which is

arguably the most sensitive quantitative assay for low passage

DENVs Realizing that this will not be possible in most

den-gue diagnostic and research laboratories, it is recommended

that data obtained using qRT-PCR to measure infectious

DENV is interpreted with caution.

Received February 22, 2013 Accepted for publication August 13, 2013 Published online September 9, 2013.

Acknowledgments: We thank Mah Sook Yee and Tan Hwee Cheng for their expert technical assistance and Ooi Eng Eong and Subhash Vasudevan for their useful suggestions.

Financial support: This work was supported by the Duke-NUS ture Research Program funded by the Ministry of Health, Singapore Authors’ addresses: Milly M Choy and Brett R Ellis, Signature Research Program in Emerging Infectious Diseases, Duke-National University of Singapore Graduate Medical School, Singapore, E-mails: milly.choy@nus.edu.sg and brettellis@mac.com Esther M Ellis and Duane J Gubler, Signature Research Program in Emerging Infectious Diseases, Duke-National University of Singapore Graduate Medical School, Singapore, E-mails: esthermarie.ellis@gmail.com and duane gubler@duke-nus.edu.sg.

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of the dengue virus E protein J Virol 73: 2547 –2551.

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COMPARISON BETWEEN MID50 AND COPY NUMBER OF DENV 1005

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