Diagnosis of West Nile virus infections:Evaluation of different laboratory methods

BACKGROUND The diagnosis of West Nile virus(WNV)is challenging due to short-term and low-level viremia,flavivirus cross-reactivity,and long immunoglobulin M(IgM)persistence.AIM To evaluate different methods for WNV detection[reverse transcription-polymerase chain reaction(RT-PCR),IgM/IgG antibodies,IgG avidity]in serum,cerebrospinal fluid(CSF),and urine samples of patients with confirmed WNV infection.METHODS The study included patients with confirmed WNV neuroinvasive infection(n=62),asymptomatic WNV seropositive individuals(n=22),and individuals with false-positive WNV IgM antibodies(n=30).WNV RNA was detected using RT-PCR.A commercial ELISA was used to detect WNV IgM/IgG antibodies with confirmation of cross-reactive samples using a virus neutralization test(VNT).IgG-positive samples were tested for IgG avidity.RESULTS The WNV-RNA detection rates were significantly higher in the urine(54.5%)/serum(46.4%)than in CSF(32.2%).According to the sampling time,the WNV-RNA detection rates in urine collected within 7 days/8-14/≥15 days were 29.4/66.6/62.5%(P=0.042).However,these differences were not observed in the CSF.The median RT-PCR cycle threshold values were significantly lower in urine(32.5,IQR=28-34)than in CSF(34.5,IQR=33-36).The frequency of positive WNV IgM and IgG significantly differed according to the sampling time in serum but not in CSF.Positive IgM/IgG antibodies were detected in 84.3/9.3%of serum samples collected within 7 days,100/71.1%of samples collected 8-14,and 100%samples collected after≥15 days.Recent WNV infection was confirmed by low/borderline avidity index(AI)in 13.6%of asymptomatic individuals.A correlation between ELISA and AI was strong negative for IgM and strong positive for IgG.No significant correlation between ELISA IgG and VNT was found.CONCLUSION The frequency of WNV RNA and antibody detection depends on the sampling time and type of clinical samples.IgG avidity could differentiate recent WNV infections from long-persisting IgM antibodies.

Isolated Hyperacute T-Waves in West Nile Encephalitis Indicating Atypical Variant of Stress-Induced Cardiomyopathy

Several cardiac outcomes have been reported with West Nile-encephalitis;however, the underlying pathophysiology remains complex. We present a 42-year-old female, with multiple sclerosis, whose neurological symptoms and respiratory decline were finally explained by the diagnosis of West Nile-encephalitis. During her admission, the isolated peaked T-waves indicated the underlying stress-induced cardiomyopathy. The absence of all other causes of hyperacute T-waves, their subsequent resolution with the resolution of infection and improvement in wall motion abnormalities, further supported the association. This case highlights the importance of considering hyperacute T-waves in an approach towards the diagnosis of WNV-encephalitis related atypical variant of stress-induced cardiomyopathy.

Prevalence of West Nile virus in Mashhad,Iran:A population-based study

Objective:To evaluate the prevalence of West Nile virus seropositivity in the general population of Mashhad.Northeast of Iran.Methods:One hundred and eighty two individuals living in the city of Mashhad were studied using cluster sampling method.Both IgM and IgG antibodies against WNV were detected by ELISA method.Results:In this study,the overall IgG seroprevalence of positive West Nile virus was 11%;however.IgM antibody was not found in the participants.Conclusions:Our study suggested that the prevalence rate of West virus is considerable in Mashhad city.It seems necessary for clinicians and health care workers to be aware of WNV infection in the Northeast Iran.

Mutagenesis of D80-82 and G83 Residues in West Nile Virus NS2B: Effects on NS2B-NS3 Activity and Viral Replication

Flaviviral NS2B is a required cofactor for NS3 serine protease activity and plays an important role in promoting functional NS2B-NS3 protease configuration and maintaining critical interactions with protease catalysis substrates. The residues D80DDG in West Nile virus (WNV) NS2B are important for protease activity. To investigate the effects of D80DDG in NS2B on protease activity and viral replication, the negatively charged region D80DD and the conserved residue G83 of NS2B were mutated (D80DD/E80EE, D80DD/K80KK, D80DD/A80AA, G83F, G83S, G83D, G83K, and G83A), and NS3 D75A was designated as the negative control. The effects of the mutations on NS2B-NS3 activity, viral translation, and viral RNA replication were analyzed using kinetic analysis of site-directed enzymes and a transient replicon assay. All substitutions resulted in significantly decreased enzyme activity and blocked RNA replication. The negative charge of D80DD is not important for maintaining NS2B function, but side chain changes in G83 have dramatic effects on protease activity and RNA replication. These results demonstrate that NS2B is important for viral replication and that D80DD and G83 substitutions prevent replication; they will be useful for understanding the relationship between NS2B and NS3.

Development and Characterization of West Nile Virus Replicon Expressing Secreted Gaussia Luciferase

We developed a Gaussia luciferase (Gluc) reporter replicon of West Nile virus (WNV) and used it to quantify viral translation and RNA replication. The advantage of the Gluc replicon is that Gaussia luciferase is secreted into the culture medium from cells transfected with Gluc replicon RNA, and the medium can be assayed directly for luciferase activity. Using a known Flavivirus inhibitor (NITD008), we demonstrated that the Gluc-WNV replicon could be used for antiviral screening. The Gluc-WNV-Rep will be useful for research in antiviral drug development programs, as well as for studying viral replication and pathogenesis of WNV.

West Nile virus: A re-emerging pathogen revisited

West Nile virus(WNV), a flavivirus of the Flaviviridae family, is maintained in nature in an enzootic transmission cycle between avian hosts and ornithophilic mosquito vectors, although the virus occasionally infects other vertebrates. WNV causes sporadic disease outbreaks in horses and humans, which may result in febrile illness, meningitis, encephalitis and flaccid paralysis. Until recently, its medical and veterinary health concern was relatively low; however, the number, frequency and severity of outbreaks with neurological consequences in humans and horses have lately increased in Europe and the Mediterranean basin. Since its introduction in the Americas, the virus spread across the continent with worrisome consequences in bird mortality and a considerable number of outbreaks among humans and horses, which have resulted in the largest epidemics of neuroinvasive WNV disease ever documented. Surprisingly, its incidence in human and animal health is very different in Central and South America, and the reasons for it are not yet understood. Even though great advances have been obtained lately regarding WNV infection, and although efficient equine vaccines are available, no specific treatments or vaccines for humanuse are on the market. This review updates the most recent investigations in different aspects of WNV life cycle: molecular virology, transmission dynamics, host range, clinical presentations, epidemiology, ecology, diagnosis, control, and prevention, and highlights some aspects that certainly require further research.

Innate host responses to West Nile virus: Implications for central nervous system immunopathology

West Nile virus(WNV) is an emerging neurotropic flavivirus that has recently spread to America and Southern Europe via an enzootic/epizootic bird-mosquito-bird transmission cycle. The virus can occasionally infect humans through mosquito bites, and man-to-man transmission has also been reported via infected blood or organ donation. In the human host, WNV causes asymptomatic infection in about 70%-80% of cases, while < 1% of clinical cases progress to severe neuroinvasive disease; long-term neurological sequelae are common in more than 50% of these severe cases. Thepathogenesis of the neuroinvasive form of WNV infection remains incompletely understood, and risk factors for developing severe clinical illness are largely unknown. The innate immune response plays a major role in the control of WNV replication, which is supported by the fact that the virus has developed numerous mechanisms to escape the control of antiviral interferons. However, exaggerated inflammatory responses lead to pathology, mainly involving the central nervous system. This brief review presents the salient features of innate host responses, WNV immunoevasion strategies, and WNV-induced immunopathology.

MATHEMATICAL ANALYSIS OF WEST NILE VIRUS MODEL WITH DISCRETE DELAYS

The paper presents the basic model for the transmission dynamics of West Nile virus （WNV）. The model, which consists of seven mutually-exclusive compartments representing the birds and vector dynamics, has a locally-asymptotically stable disease- free equilibrium whenever the associated reproduction number （R0） is less than unity. As reveal in [3, 20], the analyses of the model show the existence of the phenomenon of backward bifurcation （where the stable disease-free equilibrium of the model co-exists with a stable endemic equilibrium when the reproduction number of the disease is less than unity）. It is shown, that the backward bifurcation phenomenon can be removed by substituting the associated standard incidence function with a mass action incidence. Analysis of the reproduction number of the model shows that, the disease will persist, whenever R0 〉 1, and increase in the length of incubation period can help reduce WNV burden in the community if a certain threshold quantities, denoted by △b and △v are negative. On the other hand, increasing the length of the incubation period increases disease burden if △b 〉 0 and △v 〉 0. Furthermore, it is shown that adding time delay to the corresponding autonomous model with standard incidence （considered in [2]） does not alter the qualitative dynamics of the autonomous ：system （with respect to the elimination or persistence of the disease）.

Immunogenic potential and protective efficacy of formalin inactivated circulating Indian strain of West Nile virus

Objective:To assess the best suitable condition for virus inactivation.and to study the immunogenic potential and protective efficacy of a circulating West Nile virus(WNV) strain in Assam.Methods:Bulk preparation of circulating WNV:WNIRGC07(GeneBank ID:HQ246154).was undertaken in a bioreaclor using eytodex-1.Virus Inactivation was done in three different conditions:22 ℃.4 ℃ and room temperature.The virus preparations were evaluated for antigenicity by ELISA and toxicity by cell proliferation kit.Virus efficacy was done in-viro on swiss albino mice against standard Indian WNV and Japanese encephalitis virus(JKV)strain.Humoral and cell mediated immune response was evaluated in mice sera by ELISA and neutralization assay.Results:Inactivation at 22 ℃ was found to be more suitable in terms of less toxicity and high antigenicity.The same was selected to study the immune response and efficacy in mice.It induced neutralizing antibody titre of 1:625 and high EgG response.In vivo experiment showed 100% protective efficacy against WNV and 20.8% cross protective efficacy against JEV.Further assessment of cellular immunity through immunized mice revealed augmentation of high levels of pro-inflammatory cytokines and moderate levels of anti—cytokines indicating a mixed balance of Th1 and Th2 response.Conclusions:Findings suggest that formalin inactivated Indian WNV strain has a good immunogenic potential.This is the first study on assessment of immunogenic potential of a lineage 5 strain of WNV.Our study reveals that it would be a promising and effective candidate for vaccine studies which warrants further evaluation.

Persistent delayed auditory memory and executive function deficits 5 years after West Nile Virus Encephalitis: A neuropsychological and neuroimaging single case study

There are presently no in-depth published neuropsychological studies of West Nile Virus (WNV) encephalitis patients that have been well-correlated with high resolution structural MRI. In this study a middleaged male who developed West Nile Virus encephalitis five years previously was examined three times over a two year period. We examined him with the Wechsler Adult Intelligence Scales—Fourth Edition and the Wechsler Memory Scale—Fourth Edition (WAIS-IV/WMS-IV) and Advanced Clinical Solutions battery supplemented by tests of attention, executive, motor and sensory functions. Neuroradiological imaging revealed hypodensities within the left hippocampus in the axial and coronal planes with T2-FLAIR MRI. The man was previously high functioning and although he had prior history of well-controlled epilepsy it seems unlikely that the epilepsy could fully account for the neuropathological changes. The patient had previously completed a demanding six year double science degree program before he became ill with WNV and he had been a successful manager and director of a research company. Delayed auditory memory scores were at least two standard deviation units below age expected levels and semantic fluency and Booklet Category Tests of executive function were also in the impaired range. Moreover the illness onset profile of muscle weakness, extreme fatigue, memory complaints as well as inability to carry out research projects involving planning on the job were highly consistent with WNV encephalitis. If the memory and executive function deficits had been premorbid manifestations of epilepsy it is unlikely he would have attained the levels he did educationally and occupationally. This left hippocampal lesion is characteristic of other encephalitic viral infections such as herpes simplex virus. To our knowledge this is the first lateralized WNV encephalitis medial temporal lobe patient in the published literature.

Review on Infections of the Central Nervous System by St. Louis Encephalitis, Rocio and West Nile Flaviviruses in Brazil, 2004-2014

Rocio (ROCV), Saint Louis encephalitis (SLEV) and West Nile (WNV) are Flavivirus (Flaviviridae) probably carried by birds and transmitted by Culex mosquitoes. We show here a review on infections of the central nervous system by St. Louis Encephalitis, Rocio and West Nile Flaviviruses in Brazil, 2004-2014. In the last 10 years, serologic surveys in horses showed high proportions of seropositive animals which point out that SLEV and ROCV have circulated infecting horses in west-central, southeast and other regions of Brazil and that WNV has been introduced into Brazil and circulates mostly in Pantanal region. However humans infected by WNV have not been reported. In the State of Sao Paulo: SLEV was isolated from a case clinically diagnosed as dengue in 2004;in 2006, 6 SLEV patients including 3 cases of menigoencephalitis were reported in the middle of a large epidemic of dengue type 3;and in 2008, 1 patient with acute febrile illness that was IgM-positive for dengue was found infected by SLEV by detection of the virus genome. In 2010, ROCV genome was detected in the cerebrospinal fluids of 2 patients from the northern region with meningoenchephalitis and also AIDS. This was the first report of infections by ROCV in the last 34 years and curiously, it occured more than 2000 km from where the virus was firstly found. It is necessary to improve the surveillance of SLEV, ROCV and WNV in Brazil.

From <i>Culex</i>Exposure to West Nile Virus Infection: Screening of Specific Biomarkers

West Nile virus (WNV) is a mosquito-borne flavivirus contributing yearly, to birds, horses and human morbidity and mortality throughout the world. WNV is transmitted mainly by mosquitoes, predominantly by Culex species, to avian hosts and other vertebrates. Since the mid-1990s, WNV outbreaks and severe human cases (i.e., West Nile neuroinvasive disease) have increased throughout the North hemisphere. The absence of human vaccine and effective therapy needs to understand the pathogenesis of WN severe disease as well as factors participating in WNV transmission and mosquito exposure. The exploration of the host/vector interaction at the individual level using host antibody response against mosquito salivary proteins has open news research opportunities aiming to increase the impact of surveillance and WNV vector control strategies. This review describes Culex saliva specific biomarkers as a helpful tool to estimate exposure to vector bites and risk for WNV infection, summarizes recent advances regarding WNV vector control strategies and highlights potential specific biomarkers of WN disease severity.

Cauda equina arachnoiditis–a rare manifestation of West Nile virus neuroinvasive disease:A case report

BACKGROUND Data regarding the neuroradiology features of the West Nile virus neuroinvasive disease(WNV NID)is rather scarce.To contribute to the knowledge of the WNV NID,we present a patient with a combination of encephalitis and acute flaccid paresis,with cauda equina arachnoiditis as the main magnetic resonance(MR)finding.CASE SUMMARY A 72-year-old female patient was admitted due to fever,headache and gait instability.During the first several days she developed somnolence,aphasia,urinary incontinence,constipation,and asymmetric lower extremities weakness.Cerebrospinal fluid analysis indicated encephalitis.Native brain computed tomography and MR were unremarkable,while spinal MR demonstrated cauda equina enhancement without cord lesions.Virology testing revealed WNV IgM and IgG antibodies in serum and cerebrospinal fluid,which confirmed acute WNV NID.The treatment was supportive.After two months only a slight improvement was noticed but cognitive impairment,loss of sphincter control and asymmetric inferior extremities weakness remained.The patient died after a month on chronic rehabilitation.CONCLUSION Cauda equina arachnoiditis is a rare,but possible neuroradiological feature in acute flaccid paresis form of WNV NID.

Using a Collateral Damage Model to Explain Survival Data in West Nile Virus Infections

Simulation code for a model of the adaptive immune response seen in flavivirus infections is used to explain the immunopathological consequences seen in West Nile Virus virus (WNV) infections. We use a model that specifically handles the differences in how the virus infects resting cells, the G0 state, versus dividing cells, the G1 state, which includes vastly increased MHC-I upregulation for resting cells over dividing cells. The simulation suggests how the infection progresses in a one host model and the results shed insight into the unusual survival curve data obtained for this infection: there is an increase in health even though viral load has increased.

Impact of climate change on the global circulation of West Nile virus and adaptation responses:a scoping review

Background West Nile virus(WNV),the most widely distributed flavivirus causing encephalitis globally,is a vector-borne pathogen of global importance.The changing climate is poised to reshape the landscape of various infectious diseases,particularly vector-borne ones like WNV.Understanding the anticipated geographical and range shifts in disease transmission due to climate change,alongside effective adaptation strategies,is critical for mitigating future public health impacts.This scoping review aims to consolidate evidence on the impact of climate change on WNV and to identify a spectrum of applicable adaptation strategies.Main body We systematically analyzed research articles from PubMed,Web of Science,Scopus,and EBSCOhost.Our criteria included English-language research articles published between 2007 and 2023,focusing on the impacts of climate change on WNV and related adaptation strategies.We extracted data concerning study objectives,populations,geographical focus,and specific findings.Literature was categorized into two primary themes:1)climate-WNV associations,and 2)climate change impacts on WNV transmission,providing a clear understanding.Out of 2168 articles reviewed,120 met our criteria.Most evidence originated from North America(59.2%)and Europe(28.3%),with a primary focus on human cases(31.7%).Studies on climate-WNV correlations(n=83)highlighted temperature(67.5%)as a pivotal climate factor.In the analysis of climate change impacts on WNV(n=37),most evidence suggested that climate change may affect the transmission and distribution of WNV,with the extent of the impact depending on local and regional conditions.Although few studies directly addressed the implementation of adaptation strategies for climate-induced disease transmission,the proposed strategies(n=49)fell into six categories:1)surveillance and monitoring(38.8%),2)predictive modeling(18.4%),3)cross-disciplinary collaboration(16.3%),4)environmental management(12.2%),5)public education(8.2%),and 6)health system readiness(6.1%).Additionally,we developed an accessible online platform to summarize the evidence on climate change impacts on WNV transmission(https://2xzl2o-neaop.shinyapps.io/WNVScopingReview/).Conclusions This review reveals that climate change may affect the transmission and distribution of WNV,but the literature reflects only a small share of the global WNV dynamics.There is an urgent need for adaptive responses to anticipate and respond to the climate-driven spread of WNV.Nevertheless,studies focusing on these adaptation responses are sparse compared to those examining the impacts of climate change.Further research on the impacts of climate change and adaptation strategies for vector-borne diseases,along with more comprehensive evidence synthesis,is needed to inform effective policy responses tailored to local contexts.

Modeling West Nile Virus transmission in birds and humans:Advantages of using a cellular automata approach

In Canada,the periodic circulation of West Nile Virus(WNV)is difficult to predict and,beyond climatic factors,appears to be related to the migratory movements of infected birds from the southern United States.This hypothesis has not yet been explored in a spatially distributed model.The main objective of this work was to develop a spatially explicit dynamic model for the transmission of WNV in Canada,that allows us to explore non-climate related hypotheses associated with WNV transmission.A Cellular Automata(CA)approach for multiple hosts(birds and humans)is used for a test region in eastern Ontario,Canada.The tool is designed to explore the role of host and vector spatial heterogeneity,host migration,and vector feeding preferences.We developed a spatialized compartmental SEIRDS-SEI model for WNV transmission with a study region divided into 4 rectangular cells.We used 2010–2021 bird data from the eBird project and 2010–2019 mosquito data collected by Ontario Public Health to mimic bird and mosquito seasonal variation.We considered heterogeneous bird densities(high and low suitability areas)and homogeneous mosquito and human densities.In high suitability areas for birds,we identified 5 entry points for WNV-infected birds.We compared our simulations with pools of WNV-infected field collected mosquitoes.Simulations and sensitivity analyses were performed using MATLAB software.The results showed good correspondence between simulated and observed epidemics,supporting the validity of our model assumptions and calibration.Sensitivity analysis showed that a 5%increase or decrease in each parameter of our model except for the biting rate of bird by mosquito(c^(B,M)and mosquito natural mortality rate(d^(M)),had a very limited effect on the total number of cases(newly infected birds and humans),prevalence peak,or date of occurrence.We demonstrate the utility of the CA approach for studying WNV transmission in a heterogeneous landscape with multiple hosts.

A Virus-type Specific Serological Diagnosis of Flavivirus Infection Using Virus-like Particles

Many flaviviruses are emerging and reemerging pathogens, such as West Nile virus （WNV）, dengue virus （DENV）, yellow fever virus （YFV）, and Japanese encephalitis virus. Serological assay is the dominant method for diagnosis of flavivirus infections in human. Because antibodies generated during flavivirus infections cross-react with other flavivirus members, plaque reduction neutralization test （PRNT） is the only available assay to determine the infecting flavivirus type. Since PRNT requires culturing raw viruses, it must be performed in biosafety levet-3 or level-4 containment for many flaviviruses, and takes more than ten days to complete. To overcome these problems, we have developed flavivirus viral-like particles （VLPs） that could be used to replace raw viruses in the neutralization assay. The VLPs were prepared by trans packaging a luciferase-reporting replicon with viral structural proteins. This novel assay involves three simple steps： （i） VLPs from a panel of flaviviruses are incubated with flavivirus-infected sera at 37℃ for 1 h; （ii）the neutralized VLPs are used to infect Vero cells; and （iii） the infected cells are measured for luciferase activities at 22 h post-infection. The virus type whose VLP is most efficiently neutralized by the serum specimen （as quantified by the luciferase activities） is the etiologic agent. As a proof-of-concept, we show that a WNV-infected mouse serum neutralized the WNV VLP more efficiently and selectively than the DENV and YFV VLPs. Our results demonstrate that the VLP neutralization assay maintains the ＂gold standard＂ of the classic PRNT; importantly, it shortens the assay time from 〉10 days to 〈1 day, and can be performed in biosafety level-2 facility.

Regulation of cell survival and death during Flavivirus infections

Flaviviruses, ss(+) RNA viruses, include many of mankind's most important pathogens. Their pathogenicity derives from their ability to infect many types of cells including neurons, to replicate, and eventually to kill the cells. Flaviviruses can activate tumor necrosis factor α and both intrinsic(Bax-mediated) and extrinsic pathways to apoptosis. Thus they can use many approaches for activating these pathways. Infection can lead to necrosis if viral load is extremely high or to other types of cell death if routes to apoptosis are blocked. Dengue and Japanese Encephalitis Virus can also activate autophagy. In this case the autophagy temporarily spares the infected cell, allowing a longer period of reproduction for the virus, and the autophagy further protects the cell against other stresses such as those caused by reactive oxygen species. Several of the viral proteins have been shown to induce apoptosis or autophagy on their own, independent of the presence of other viral proteins. Given the versatility of these viruses to adapt to and manipulate the metabolism, and thus to control the survival of, the infected cells, we need to understand much better how the specific viral proteins affect the pathways to apoptosis and autophagy. Only in this manner will we be able to minimize the pathology that they cause.

Impacts of Increasing Temperature on the Future Incidence of West Nile Neuroinvasive Disease in the United States

Multiple studies have identified links between climate and West Nile virus disease since the virus arrived in North America. Here we sought to extend these results by developing a Health Impact Function (HIF) to generate county-level estimates of the expected annual number of West Nile neuroinvasive disease (WNND) cases based on the county’s historical WNND incidence, annual average temperature, and population size. To better understand the potential impact of projected temperature change on WNND risk, we used the HIF to project the change in expected annual number of WNND cases attributable to changing temperatures by 2050 and by 2090 using data from five global climate models under two representative concentration pathways (RCP4.5 and RCP8.5). To estimate the costs of anticipated changes, as well as to enable comparisons with other public health impacts, projected WNND cases were allocated to nonfatal and fatal outcomes, then monetized using a cost-of-illness estimate and the U.S. Environmental Protection Agency’s value of a statistical life, respectively. We found that projected future temperature and population changes could increase the expected annual number of WNND cases to ≈2000 - 2200 cases by 2050 and to ≈2700 - 4300 cases by 2090, from a baseline of 970 cases. Holding population constant at future levels while varying temperature from a 1995 baseline, we estimated projected temperature change alone is responsible for ≈590 and ≈960 incremental WNND cases in 2050 and 2090 (respectively) under the RCP4.5 scenario, and ≈820 and ≈2500 cases in 2050 and 2090 (respectively) for the RCP8.5 scenario, with substantial regional variation. The monetized impact of these temperature-attributable incremental cases is estimated at $0.5 billion in 2050 and $1.0 billion in 2090 under the RCP4.5 scenario, and $0.7 billion in 2050 and $2.6 billion in 2090 under the RCP8.5 scenario (undiscounted 2015 U.S. dollars).

A Simulation for Flavivirus Infection Decoy Responses

In this work, we discuss the development of simulation code for a model of the cross-reactive adaptive immune response seen in flavivirus infections. The model specifically addresses flavivirus pathogen virulence in G0?vs G1?cell states. The MHC-I upregulation of resting cells (G0 state) allows the T-cells generated for flavivirus peptide antigens to attack healthy cells also. The cells in G1?state are not upregulated as much and so virus hides in them and hence is propagated upon rupture. Hence, this type of model is referred to as a decoy model because the immune system is decoyed into preferentially recognizing the upregulated cells while the virus actively propagates in another small, but important, cell population. We show that the generic assumption of upregulation via a model which includes the?G0/G1?differential upregulation leads to immunopathological consequences. We outline the details behind the simulation code decisions and provide some theoretical justification for our model of collateral damage and upregulation.