Avahan, the India AIDS Initiative, delivers HIV prevention services to high-risk populations at scale. Although the broad costs of such HIV interventions are known, to-date there has been little data available on the comparative costs of reaching different target groups, including female sex workers (FSWs), replace with ‘high risk men who have sex with men (HR-MSM) and trans-genders.Costs are estimated for the first three years of Avahan scale up differentiated by typology of female sex workers , HR-MSM and transgenders in urban districts in India: Mumbai and Thane in Maharashtra and Bangalore in Karnataka. Financial and economic costs were collected prospectively from a provider perspective. Outputs were measured using data collected by the Avahan programme. Costs are presented in US$2008.Costs were found to vary substantially by target group. Non-governmental organisations (NGOs) working with transgender populations had a higher mean cost (US $116) per person reached compared to those dealing primarily with FSWs (US $75-96) and MSWs (US $90) by the end of year three of the programme in Mumbai. The mean cost of delivering the intervention to HR-MSMs (US $42) was higher than delivering it to FSWs (US $37) in Bangalore. The package of services delivered to each target group was similar, and our results suggest that cost variation is related to the target population size, the intensity of the programme (in terms of number of contacts made per year) and a number of specific issues related to each target group.Based on our data policy makers and program managers need to consider the ease of accessing high risk population when planning and budgeting for HIV prevention services for these populations and avoid funding programmes on the basis of target population size alone. It is estimated that around 2.5 million people were living with HIV/AIDS in India in 2006 -3. Much Avahan delivers HIV prevention services to a wide range of high risk populations. Female sex workers (FSW) are the largest group, with Avahan targeting an estimated 310,000 FSWs. In India, FSWs are categorized into different typologies, based on where they recruit or solicit clients and not where they live or entertain the clients ,7. The mThere are a limited number of peer reviewed studies on the costs of HIV prevention services in Asia -14. ThesAs a part of the overall evaluation plan, Avahan was subject to an intensive costing effort and an economic evaluation in four southern states, Karnataka, Maharashtra, Andhra Pradesh and Tamil Nadu during 2005-2008. This evaluation covers over eighty districts, and thus provides an opportunity to understand the drivers of HIV prevention costs . This paThis study presents the costs of HIV prevention in two large scale urban settings, Mumbai and Thane in Maharashtra and Bangalore in Karnataka. In Mumbai and Thane, we measured the costs of delivering HIV prevention to different typologies of FSWs, HR-MSM and transgenders. A cross sectional behavioural and biological survey conducted in April 2006 in Maharashtra found an HIV prevalence of 28.1% (22.2-34.8) among brothel-based FSWs and 19.2%(13.7-26.2) among street-based FSWs [In Mumbai/Thane, Avahan funds 16 separate non-governmental organizations (NGOs) to deliver HIV prevention services. Each of these NGOs targets different high risk populations . For each NGO, data was collected on project activities, financial expenditure and outputs. Time-sheets were used to determine allocation of resources between different activities and population groups. For all the other NGOs studied, data was collected from their routine reports and no fieldwork was done. Expenditure data was obtained from routine financial and management reporting, staff records and interviews with staff. We estimate both financial and economic costs. Financial costs represent the money spent by the programme to deliver the intervention, whereas economic cost includes the value of all inputs . Therefore, for the detailed costing sites, data on donated goods and services were also collected from the programme. The economic costs of these items were valued at market prices obtained from local shops and interviews with project staff.3% was used. Recurrent costs include all personnel costs, travel, building operating and maintenance supplies, cost of condoms, medical supplies and all other supplies costs.Costs were classified according to three characteristics: the phase of implementation, organisational level where costs are incurred and type of cost. The time period between the decision to implement an intervention and starting its delivery to the beneficiaries was defined as the start up phase. All costs incurred in the start-up period were annualised to reflect utility beyond the start up period. All costs incurred after the start up period were defined as implementation costs. Costs were collected both from the state level (supporting the NGOs) and from each NGO. Costs are categorised as either recurrent or capital costs using a definition of capital cost to be an item with a useful life of more than one year. Capital costs include equipment, furniture and fixtures, vehicles, rental deposits and start up costs. Capital equipment was assumed to have a life of between 5 and 10 years, depending on the item. Capital costs were annualised to reflect the utility of their use during the course of the programme. A discount rate of At the start of an intervention in a district or sub-district, NGOs conducted a formal external mapping and size estimation exercise. Some state-level lead implementing partners updated these numbers on a regular basis (every 12 to 18 months) using programme data- others conducted formal size estimation exercises; others used programme data . ProgramSince the cost estimates cover more than one year of expenditure, where relevant, costs have been adjusted to US$2008 using the Gross Domestic Product (GDP) deflator reported by the Indian Ministry of Finance . FurtherThe estimated target population, population reached, contacts made and clinic visits per year by typology of high-risk population are presented in Table Table Figure The unit costs by typology (estimates made from detailed costing sites/ or single target population sites only) are shown in Table There is considerable interest by the Government of India, and other countries to sustain HIV prevention programmes, but their cost remains a concern. Improving the knowledge of factors that drive costs can assist programme budgeting and help in deciding the optimal resource allocation required to deliver these programmes. Previous resource estimates have tended to deal with financial costs only and assumed that they were linear across population typologies . Many goWe find that the costs of reaching different populations with HIV prevention interventions in the similar settings vary substantially. The costs for all typologies are at the higher end of those found in previous studies in India of both Avahan and HIV prevention delivered by others ,14-17. TThe factors that drive cost differences between high-risk population groups are complex. As with between settings, unit cost variation within similar settings is likely to be related to the size of the target group. This is demonstrated by the decreases in unit costs over time as the programme expanded Table . MoreoveThe scale effect within NGO sites for different target groups is also illustrated by our cost breakdowns. Our cost breakdowns show that the proportion of both capital and personnel related costs are substantially higher per person reached in smaller target groups whereas other costs such as those for STI supplies (as a proportion of unit costs) remain more uniform across different target groups . This is likely to be due, in part, to the fact that each NGO needs a certain level of fixed capacity in key areas, such as support and supervision for outreach workers. Planners and funders therefore need to consider whether it is worth encouraging NGOs targeting smaller groups to share these fixed costs between one another, and explore how their funding mechanisms can better encourage the more efficient use of fixed resources.Our data also suggest that higher intensity of service, in terms of numbers of contacts made, is associated with higher unit cost per person reached, albeit in a very limited way. In Mumbai/ Thane the magnitude of the difference in intensity (ranging from 4.2 to 6.6 contacts per year) is aligned to magnitude of the cost differences observed, with the exception of street–based FSWs, particularly in year 2. In year three there were specific issues with the management of the Hirja programme that meant that costs remained high, despite a lower intensity of effort. In Bangalore however, this is much less the case, as differences in intensity are much lower, and thus overall we found no statistically significant relationship between intensity and cost. More work needs to be done with large sample sizes to explore this relationship further.We observe little variation in the proportion of activity costs between different population groups. This indicates that no group required a special mix of activities, but nevertheless, when asked to interpret our findings programme staff identified some specific issues when working with the non-fixed location groups, such as hijras. For example, it took longer for staff to orientate themselves regarding the nature of the hijra population. Moreover, it took time to build rapport with the hijra population; requiring a higher frequency of visits from the NGO staff compared than other groups. Programme staff also highlighted issues with keeping track of street -based FSWs repeatedly, due to the highly mobile nature of that population. In addition, street-based FSWs were considered more reluctant to participate in the project services, because of the time commitment and the corresponding loss of clients and income. Street- based FSWs were also worried that visiting drop-in centres would reveal their identity and lead to more stigma and harassment from police and the local community. The factors were thought by the NGOs to increase the level of outreach and thus cost of reaching these groups.This study is limited by various factors, the most important of which is its small sample size. Ideally when exploring cost differences between different groups one would use statistical techniques, examining costs and cost drivers over a large number of sites. However, even in such large scale settings, it is difficult to capture the detailed cost data required from a sufficient number of NGOs to enable this analysis. We are planning a follow-up econometric of costs drivers’ analysis of all Avahan sites, but as few sites target specific high risk sub-populations, there are an insufficient number of sites to explore the full impact of typology on cost statistically. We therefore needed to rely on the descriptive analysis presented above. An alternative method would be to measure costs at the client/ individual population level, and it is recommended that future studies explore opportunities to do this.Furthermore, our costs are also likely to be impacted by a number of other factors beyond target population. While the settings were similar in terms of NGO characteristics and HIV prevalence, other factors related to implementation are likely to also have an impact on costs. For example the programme had some difficult phases in particular districts in Mumbai due to a major bomb blast in the metro and combing operations by the police. There was sudden closure of bars due to government instruction and the programme had to change the strategy to reach the sex workers in the place of residence. The issue of frequent raids in brothels also affected programme services in certain areas. Again this limits the robustness of results, even though most of the above effects were temporary in nature.Our findings suggest that policy makers, planners and analysts should consider the typology of the target population when conducting efficiency analyses and setting budgets across HIV prevention programmes. Analytically, care should be taken to judge costs and efficiency in the context of the populations they service. However, setting budgets using a fixed amount per person reached risks penalising those NGOs who are targeting more difficult to reach groups and may create a perverse incentive to focus on high risk groups that cost less to reach.Different HIV prevention target groups present multiple issues in delivery of services and interventions, reflected in the cost variation. Policy makers and programme managers are therefore recommended to examine the particular circumstances of the populations being reached when setting budgetary limits for HIV prevention services for high risk groups.The authors declare that they have no competing interests.SC: contributed to the design, data collection, analysis, interpretation and prepared the first draft of the paper; AV: contributed to the analysis, interpretation of the data and the manuscript; PV: contributed to manuscript preparation; BR and GY: assisted in the data collection, data entry, preliminary data analysis and generation of tables; MA: Principal Investigator of the main study and contributed to the design of the study.This research was funded by the Bill & Melinda Gates Foundation and also financial support from HIV Research Trust UK.


The genome of a novel virus of the house cricket consists of a 2,517-nucleotide (nt) circular single-stranded DNA (ssDNA) molecule with 4 open reading frames (ORFs). One ORF had a low identity to circovirus nucleotide sequences (NS). The unique properties of this volvovirus suggested that it belongs to a new virus family or genus. Acheta domesticus, is highly susceptible to a densovirus, Acheta domesticus densovirus (AdDNV), which has caused severe outbreaks since September 2009 and decimated A. domesticus stocks in North America. Samples received from die-offs were invariably positive for this virus. However, some recently received samples from mass cricket die-offs in North America were negative for AdDNV.Cricket-breeding facilities in the United States produce billions of pet-feeder crickets annually , 2. The AdDNV-negative crickets (20 g) were homogenized in 20 ml of a 3:1 mixture of phosphate-buffered saline (PBS) and carbon tetrachloride. After low-speed centrifugation, the upper aqueous phase was passed through 0.45-nm filters and putative viruses were pelleted by centrifugation for 1.5 h at 40,000 rpm and resuspended in a small volume of Tris-EDTA (TE) buffer followed by DNase A and RNase A treatments to remove contaminating nucleic acids. Electron microscopy examination of a 100-fold dilution of the resuspended pellet revealed highly concentrated icosahedral particles of about 18 nm in diameter.Acheta domesticus volvovirus  was proposed.DNA extracted from purified virus by the High Pure viral nucleic acid kit (Roche Applied Science) was resistant to restriction endonucleases and presumably single stranded. Native viral DNA was used for double-stranded DNA synthesis at 30°C by ϕ29 DNA polymerase . The seqNumbering of the genome started with the putative nonanucleotide origin of replication (1-TAGTATTAC), located, as for circo- or cycloviruses , betweenThe lack of sequence identity and the differences in genome organization and size indicated a new virus family or genus. To our knowledge, this is the first circular single-stranded DNA virus in insects that is not related to cycloviruses , 8, circKC543331.The GenBank accession number for AdVVV is


Fusellovirus-Sulfolobus system. The host, Sulfolobus, is a hyperthermophilic acidophile endemic to sulfuric hot springs worldwide. The Fuselloviruses, also known as Sulfolobus Spindle-shaped Viruses (SSVs), are “lemon” or “spindle”-shaped double-stranded DNA viruses, which are also found worldwide. Although a few studies have addressed the host-range for the type virus, Sulfolobus Spindle-shaped Virus 1 (SSV1), using common Sulfolobus strains, a comprehensive host-range study for SSV-Sulfolobus systems has not been performed. Herein, we examine six bona fide SSV strains  and their respective infection characteristics on multiple hosts from the family Sulfolobaceae. A spot-on-lawn or “halo” assay was employed to determine SSV infectivity (and host susceptibility) in parallel challenges of multiple SSVs on a lawn of a single Sulfolobus strain. Different SSVs have different host-ranges with SSV1 exhibiting the narrowest host-range and SSVRH exhibiting the broadest host range. In contrast to previous reports, SSVs can infect hosts beyond the genus Sulfolobus. Furthermore, geography does not appear to be a reliable predictor of Sulfolobus susceptibility to infection by any given SSV. The ability for SSVs to infect susceptible Sulfolobus host does not appear to change between 65°C and 88°C ; however, very low pH appears to influence infection. Lastly, for the virus-host pairs tested the Fusellovirus-Sulfolobus system appears to exhibit host-advantage. This work provides a foundation for understanding Fusellovirus biology and virus-host coevolution in extreme ecosystems.An emerging model for investigating virus-host interactions in hyperthermophilic Archaea is the  Fuselloviridae and their hyperthermophilic archaeal hosts , isolated from the host Sulfolobus shibatae strain B12 from geothermal springs in Beppu, Japan ] and are found either episomally, integrated in the host genome as proviruses, or packaged as fully-assembled virions. To date, eleven “free” fuselloviruses have been reported in the literature: SSV1 (a.k.a. SAV1)  but not Sulfolobus acidocaldarius , infection has only been shown for S. solfataricus strain P2 ; SSV2 ; SSV3 ; SSVK1 ; SSVRH ; and, a recent isolate, SSVL1 . These SSVs along with a set of 13 Sulfolobaceae were used to complete a comprehensive host range study, which includes a simple test to determine if SSVs infect beyond the genus Sulfolobus. To evaluate the stability of SSV infectivity under variations in environmental conditions that might be encountered in natural habitats, assays were also performed with varying temperature and pH.Because Sulfolobus strains stored at −80°C were partially thawed on ice. 10–20 μL of stock was added to a flask with 30 mL of media and culture was placed in a shaking water bath set at 78–80°C/70 RPM. Yeast-Sucrose (YS) media (1X) was used for most inoculations; however, for strains that do not grow well in YS media a tryptone-based media (1X) was used instead  between 0.4 and 0.6, 3.0 mL of cell suspension was used to inoculate 300 mL of pre-heated fresh medium and cultures were, again, incubated at 78–80°C/70 RPM until reaching an OD600 = 0.4–0.6, at which time virus was harvested. Harvesting virus consisted of centrifuging all 300 mL of the culture for 20 min at 6000 RPM . The supernatant was decanted and filtered through a 0.45 μm filter and 300 mL of filtrate was concentrated to ~1.5 mL using a Centricon Plus-70 spin concentration tube . Transmission electron microscopy (TEM) was used to confirm the presence of SSV-like virions (described below). Virus concentrates were stored at 4°C and used within 2–3 weeks of harvesting.Glycerol stocks of SSV-infected 600 = 0.4–0.6 culture was added to 4.5 mL of 0.25% w/v Gelrite®  in YS medium at 78°C. The mixture was spread on pre-warmed 1% w/v Gelrite plates, allowed to solidify for 15 min at RT, then incubated for 20 min at 78°C. After this short incubation period, 1.0 μL of virus suspension was spotted onto the lawns and plates were incubated for 3–9 days at 78°C . Plates were checked daily for up to 9 days. Inhibition of host cell growth, as evidenced by a visible halo of growth inhibition, was scored as a successful infection. Triton X-100 (0.01% v/v) was used as a positive control and sterile water as a negative control.Infectivity and susceptibility assays were performed essentially as in  uranyl acetate for 30 s. The stain was wicked off and the sample was air dried. Grids were imaged in a Hitachi H-7100 TEM at 75 kV. Virus images were captured at 60,000–150,000× magnification. Note that these preparations were spin-filtered and concentrated viral suspensions, not scraped plaques.2O prior to ethanol dehydration. An aliquot of cell suspension was placed on a 0.6 mm isopore membrane filter using a syringe and then dehydrated using a graded ethanol series of 35, 50, 70, 90, 95, and 100% (v/v). This dehydration step was repeated once. Following the second 100% ethanol step, the filter was placed in 100% hexamethyldisilazane (HMDS) for 30 min then air-dried. The filter was sputter-coated with gold/palladium and imaged in a Hitachi S-4700 scanning electron microscope.Cell suspensions were centrifuged (4000 RPM for 15 min), the supernatant was decanted, the cell pellet was resuspended in sodium cacodylate buffer (pH = 6) with 5% (v/v) EM-grade glutaraldehyde, and the suspension was left overnight at 4°C. Fixed cells were washed in distilled water and fixed in 2% osmium tetroxide for 2 h at 4°C, washed twice in distilled water, and resuspended in dH++ and Ca++ ions produced slightly more stable plates at low pH but, for most plates, there were sections that prematurely dissolved or that could otherwise not be scored. Low pH assays were performed until a minimum number of replicates for each host-virus pairing could be scored. Gelrite®-plates at pH > 2.0 typically maintain soft-layer and base integrity for up to 9 days at 78–80°C. Separating stacked plates with rubber risers improved stability by distributing hot air flow.For some virus-host pairings, halos were very faint and could only be viewed by holding the plate in front of and close to a light source. Flash photography of faint halos proved difficult. SSV1 and SSVL1 in particular produced faint halos. In some cases, host growth was not robust; this also resulted in faint halos. Some lawns were damaged on one section of the plate, typically by condensation. In these instances the section of the plate that could be scored was scored. The damaged section of the plate was excluded from analyses. This issue was particularly problematic in the low pH (pH < 2.0) trials. Adding additional MgSulfolobus strains have been identified by their ability to form halos of growth inhibition or plaques on lawns of virus-free isolates of S. solfataricus strains P1 and/or P2, or closely-related “S. islandicus” strains, or by direct observation of culture supernatants  for all six SSV strains tested . Differences in halo size were found to be a function of spotted virus concentration (data not shown). SSV strain-specific halo morphologies persisted regardless of the concentration of the viral suspension used. Sulfolobus-SSV cell-virus interaction was confirmed using SEM and TEM  plus the closely-related crenarchaeon Sulphurisphaera ohwakuensis. Susceptibility to SSV infection for each potential host varied. Some were susceptible to all six SSVs tested  also was susceptibile to infection by all SSVs tested , and a Icelandic strain, “S. islandicus” REN1H1 which harbors plasmids pRN1 and pRN2  relatedness. Both susceptible and completely resistant strains are in closely-related clades in Sulfolobaceae. For example, Sulfolobus tokodaii and Sulfurisphaera ohwakuensis have identical 16S rDNA genes (>99%) and S. tokodaii and S. acidocaldarius have 90% 16S rDNA genes, yet the latter pair has the same lack of susceptibility to SSV infection . However, not all of these strains were isolated from environments with these conditions. Moreover, hydrothermal environments are chronically variable. SSV1 is reported to maintain activity for at least 45 min at 89°C and pH 5.5; however, the SSV1 genome is reported to be unstable at low pH, even though the virions appear to remain intact  but neither incubation at low temperature (65°C) nor at high temperature (88°C) qualitatively changed infectivity relative to the control (78°C). These data suggest that qualitative SSV infectivity and host susceptibility is stable at different temperatures. Separate preparations of media and plates with pH values of 1.2, 2.2, 3.2, and 4.2 were then used to culture cells and grow lawns of S. solfataricus Gθ. All plates were incubated at 78–80°C. After three trials (each in triplicate), all six SSVs readily inhibited S. solfataricus Gθ growth on plates prepared at pH = 3.2 and 4.2 ; and an isolate from the Mutnovsky Volcano region (Russia). Nevertheless, other isolates from each of these regions were at least partially susceptible to a set of allopatric SSVs. In addition, an isolate from Lassen Volcanic National Park was found to be susceptible to all SSVs tested. In summary, hosts from all geographic regions tested  included at least one strain that was completely resistant to all six SSVs tested as well as some that were sensitive to infection by at least one SSV. These susceptibility data are unexpected due to phylogenetic data on Sulfolobus, which suggest that host genetic divergence is positively correlated with geographic distance between natural habitats  and “foreign vs. local” (multiple viruses on a single host) criteria for virus local adaptation  In spot on lawn assays different SSVs elicit different halo morphologies during replication; (3) Variations in both pH and host (similar to those found in environments in which SSVs are found) have qualitative effects on SSV infection but temperature does not; (4) SSV1 exhibits the narrowest host range of all SSVs tested and SSVRH has the broadest host range; (5) SSV infectivity and Sulfolobus susceptibility are independent of the geographic regions from which the hosts and viruses were isolated. These results form a solid foundation to support future work focused on determining both the genetic and molecular basis for these host-range differences as well as also for quantitative studies of local adaptation in this tractable model system.Our results indicate that: (1) Different SSV strains have different host ranges, at least three of which extend beyond the genus The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In the most basic scenario, the model suggests that R0 is maximized if a single virus particle is transferred per synapse. R0 decreases and the infection eventually cannot be maintained for larger numbers of transferred viruses, because multiple infection of the same cell wastes viruses that could otherwise enter uninfected cells. To explain the relatively large number of HIV copies transferred per synapse, we consider additional biological assumptions under which an intermediate number of viruses transferred per synapse could maximize R0. These include an increased burst size in multiply infected cells, the saturation of anti-viral factors upon infection of cells, and rate limiting steps during the process of synapse formation.HIV can spread through its target cell population either via cell-free transmission, or by cell-to-cell transmission, presumably through virological synapses. Synaptic transmission entails the transfer of tens to hundreds of viruses per synapse, a fraction of which successfully integrate into the target cell genome. It is currently not understood how synaptic transmission affects viral fitness. Using a mathematical model, we investigate how different synaptic transmission strategies, defined by the number of viruses passed per synapse, influence the basic reproductive ratio of the virus, R The rate of viral spread through the target cell population has been shown to influence the level of virus control and the pattern of disease progression The occurrence of synaptic transmission in HIV infection brings up an evolutionary question. What is the optimal number of viruses transferred from a source cell to a target cell such that the rate of viral spread is maximized? Along similar lines, how does this optimum depend on the biological assumptions? These questions are investigated here with a new mathematical model that takes into account synaptic transmission of the virus. We vary the average number of viruses transferred through a synapse and investigate how this affects the basic reproductive ratio of the virus, a measure which quantifies how fast the virus spreads through its population of target cells. The basic reproductive ratio of the virus is shown to correlate with viral fitness. For simplicity, we will refer to the number of viruses transferred per synapse as the “viral strategy” in the rest of this paper.2 viruses per synapse, typically observed in HIV infection In the simplest setting, the model gives rise to the surprising result that the optimal viral strategy is to transfer a single virus particle per synapse. Increasing the number of viruses transferred per synapse leads to a reduced basic reproductive ratio and to extinction of the infection. We subsequently examine conditions that could account for the emergence of synaptic transmission strategies that transfer of the order of 10We consider a modeling framework that builds on ordinary differential equations of virus dynamics 0x denotes uninfected cells, v denotes is the population of free virus. We assume that infected cells can be simultaneously infected by several copies of  viruses. The variable ix denotes the number of cells infected with i viruses, where the index i runs from 1 to N, the maximum number of viral genomes incorporated in a cell’s genome . Target cell production and death rates are given by λ and d. Infected cells die with a rate (i)a. It is assumed that a fraction of the viruses produced by a cell is transmitted via the free-virus pathway. The remaining fraction is transmitted via the synaptic pathway. The free-virus pathway is represented by terms multiplying parameter u. The cell-cell transmission pathway is represented by terms multiplying m to successfully transmit j copies per synapse. For convenience, all the parameters are summarized in Here In our model we used the superscripts in parenthesis to indicate the explicit dependence of certain parameters on the multiplicity of infection. For example, in general, the parameter Both systems (1) and (2) represent the overall process of infection, see i(m)}{q, which reflects the probability for an infected cell of multiplicity m to attempt to transfer i viral particles per synapse. The rate of synaptic viral transfer is given by s the mean number of viral particles that a source cell attempts to transmit to its target. The parameter m to successfully transfer j viruses to a target cell per synapse) depends on the cell’s strategy and also on the probability for an individual transferred virus particle to survive and successfully infect a target cell, which is denoted by r. An example of the probability distribution m, and for different values of r is given in s viruses to the target cells with probability 0 viruses with probability s completely characterizes the strategy. Therefore, in the rest of the paper we will simply refer to different strategies by the corresponding number, s. Using this simplification allows us to gain analytical insights when examining the costs and benefits of the different transmission strategies. In the In general, we define a viral synaptic strategy as a probability distribution, s. Our main goal is to explore how changing the number of viruses transferred per synapse (strategy s) affects the viral fitness measured by Using these modeling approaches, we can relate the important parameters of infection dynamics, such as the infectivity We start by considering a simplified version of model (1) that only takes into account synaptic transmission. We examine the effect of varying the number of viruses transferred per synapse (the strategy) on the basic reproductive ratio of the virus under different conditions. Subsequently, the full model is used to examine the synaptic transmission strategies in the context of concurrent free-virus transmission. Our base-line model makes the following assumptions:m. Thus, the mean number of virus particles that a cell produces and attempts to transmit does not depend on the multiplicity, m: The burst size of infected cells and their death rate are the same regardless of the number of resident viruses in the cell, s. Thus, if s is small, this means that a cell attempts to pass a small number of particles to many cells by forming many synapses. If s is large, then the cell’s strategy is to transfer many viral particles to a few cells, by forming few synapses, The total number of viruses that a cell will transmit via synapses during its life-span is independent of the strategy 0R. It denotes the average number of newly infected cells generated by a single infected cell at the beginning of the infection, and needs to be greater than one for the virus population to become established 0R as a function of the probability r that individual viruses successfully infect a cell following entry. Different curves are shown for different values of s (i.e. the number of viruses transferred per synapse). As expected, 0R increases for higher values of r. However, an increase in the number of transferred viruses, s, leads to a decline in the basic reproductive ratio of the virus, which is also obvious from the expression for 0R. If the value of s lies above a threshold, the infection cannot be maintained anymore. Therefore, the most efficient strategy is the transfer of a single virus particle per synapse (s = 1), while the least efficient strategy maximizes the number of transferred viruses per synapse. Similar trends are shown when considering the equilibrium number of infected cells  is more efficient and leads to a higher basic reproductive ratio than the transfer of more viruses (high s), see r.We observe the following patterns. Let us first assume that the effect of multiple infection on the burst-size is at most additive. That is, if the multiplicity of virus in the cell increases by a factor of A, the burst size increases by a factor A or less, which corresponds to the assumption g>1 in our model). Now, the basic reproductive ratio of the virus increases towards an asymptote with the number of viruses that are transferred per synapse, s . Therefore, in order for a higher value of s to maximize the basic reproductive ratio of the virus, the effect of coinfection must be superadditive.These results can be shown by examining the basic reproductive ratio of the virus given by 0R, the advantage gained by an increased burst size of coinfected cells must outweigh the cost incurred by the amount of “wasted virus”, hence leading to faster spread. This can be achieved if the burst size of multiply infected cells grows faster than the multiplicity of infection.The intuitive reason for this result is as follows. For an intermediate number of transferred viruses to optimize r in the previous sections. It is possible that the number of inhibiting factors that can bind the virus particles is limited, and thus a synapse that sends a large number of viruses into a specific target cell has a possibility to “flood” and saturate this defense 1n immune particles are available in the cell, then the first 1n viruses will be bound to them, resulting in a low individual probability of infection per virus, r. If the number of viruses entering the cell by synapse, j viruses, In this section, we will consider the effect of saturating cellular factors that can inhibit infection of the cell to a certain degree. It has been proposed that the viruses entering cells are subject to inhibition by factors that can be considered part of innate immunity r, decays with an increasing number of viruses that are passed through the synapse, because more viruses induce more immune effectors . This is a “stealth” strategy where the virus keeps a low profile and avoids strong degrees of inhibition. Transferring more viruses per synapse, s, leads to stronger induction of anti-viral effectors and to a reduction in 0R. If the number of viruses transferred per synapse is increased even more, however, drug saturation of the virus is observed, which leads to a further rise in 0R towards a second peak for high values of s. As before, even higher numbers of viruses transferred per synapse lead to wasting of viruses through infection of already infected cells and thus to a decrease in 0R. Therefore, two “optimal” viral strategies are observed in this model: a stealth and a saturation strategy. Depending on the parameters, either could be more advantageous.The above analysis assumed that there is a fixed amount of immune effectors within the cell that are depleted in action. If intracellular immune effectors can be induced by the virus, however, we observe a slightly different pattern . This scffectors , inset. z is a parameter. Case In the models above we assumed that in cell-cell transmission, all the virus particles produced were transferred via synapses. A more realistic assumption is that formation of each synapse takes a certain amount of time before the cells separate and are able to find new partners. The process of finding a partner might also be a rate-limiting step. In a spatial setting, cells have a limited number of neighbors, and therefore spread via the virological synapse is much more resource-limited than spread via free virus (the latter can occur over longer distances). Therefore, the rate of synapse formation may not be inversely proportional to the number of viruses transferred (the strategy), but rather may have a cap, that is, the maximum intensity of synapse transmission. In this model, for relatively low amounts of transferred viruses (low s), it is not possible to form enough synapses to transfer all the viruses produced. To incorporate this effect mathematically, instead of s) may be less effective compared to strategies involving the transfer of more viruses per synapse (higher s). In general, for each given infection probability, r, there will be an intermediate optimal viral transfer strategy, s, which leads to the maximum value for 0R and the maximum number of infected cells. The reason for this is directly related to the fact that synapses cannot be formed at arbitrarily high rates. Transfer of low numbers of viruses per synapse will end up wasting a lot of viruses because not enough synapses can be formed to transfer all the viruses produced.freer. If <r, the result is the same as before: for each given infection probability, r, there may be an intermediate optimal viral transfer strategy, s, which leads to the maximum value for 0R. On the other hand, if >r, the synaptic strategy with s = 1 maximizes the basic reproductive ratio of the virus. In other words, the transfer of only one virus particle per synapse leads to the fastest rate of viral spread. Therefore, the relative infection probability characteristic of the two transmission pathways plays a deciding role. This can be influenced by the survival of the offspring virus. Thus, if offspring virus has a higher likelihood to be lost during free virus compared to synaptic transmission, then an intermediate number of viruses transferred per synapse maximizes 0R. Note, however, that the exact nature of the condition can depend on the mathematical formulation of the rate limitations during synapse formation, and is explored in general terms in the This argument, however, assumes that viruses can only be transmitted through synapses. In reality, if a cell cannot form a sufficient number of synapses during its life-span to transfer all offspring virus, this remaining offspring virus population does not have to be wasted, but can be released as cell-free virus. Therefore, we re-consider this argument in the context of the full model (1) that takes into account both transmission pathways. Let us define the probability of successful infection by free virus as 0R under different sets of assumptions. In a similar way, one can compare how synaptic and free virus transmissions contribute to the basic reproductive ratio of the virus. This can be addressed with the full model (1) taking into account both transmission pathways. The kinetic parameters of the infection process are likely different in these two modes of transmission. The viral production rate is given by the distinct parameter freek and the probability of successful infection per virus is given by So far, we have examined how different synaptic transmission strategies affect the basic reproductive ratio of the virus, and defined the optimal number of transferred viruses that maximize s. For low numbers of viruses transferred per synapse (low s), fewer synaptic connections are established during the life-span of a cell. Hence, a lower fraction of the total offspring virus produced by a cell is transferred through synapses, and a higher fraction is released into the extracellular environment. In this particular model, looking at both pathways is necessary  are not independent, and have to be taken into account when studying the efficiency of different synaptic strategies. In this context, we described a model where during the life-span of the cell, the fraction of offspring virus transferred through synapses depends on the synaptic strategy, s. It remains an additive constant, which does not influence the result of the outcome of evolutionary competition. A similar argument works for the rest of the models we considered.0R, as a measure of fitness of various synaptic strategies. Here we show that this quantity is indeed a valid measure of relative evolutionary advantage of competing traits. In order to do this, we formulate an evolutionary model where two virus strains characterized by different synaptic strategies  compete for the same target cell population. Let us suppose that there are two competing strategies, 1s and 2s, and denote by ijx the number of cells infected by i copies of 1s-virus and j copies of the 2s-virus. We further denote by m copies of 1s-virus and k copies of the 2s-virus will successfully transfer q copies of 1s-virus and p copies of the 2s-virus by means of the synaptic transfer. Then, the dynamics of synaptic transmission is described by the systemIn this paper we use the basic reproductive ratio, m copies of 1s-virus and k copies of the 2s-virus. Then its strategy mks can be given by (a) the mean strategy of the resident viruses, In order to analyze this system, we need to make further assumptions on the strategies of cells infected with two different types of virus. We considered several possible choices. Suppose that a cell is infected by 1 = 1s and 2 = 3s, in the framework of the base-line model, where the first strategy has a larger basic reproductive ratio. For the parameters chosen, a successful infection is established, where only the viruses with strategy 1 = 1s are represented. The population of cells containing 2 = 3s viruses decays to zero. The competitive exclusion shown here has been observed for all the scenarios, which include models other than the base-line model. As long as a strategy possesses a higher basic reproductive ratio, it invades and drives the weaker strategy extinct. This supports the claim that the basic reproductive ratio is an appropriate viral parameter to characterize the fitness of the strategy in the context of synaptic transmission.The result of a typical simulation is presented in Finally, we would like to discuss the general evolutionary approach we pursue in this paper and put it in a greater context. In our line of thinking we implicitly assume that if a feature is present then it must be adaptive 0R values of the virus, and say that, given enough time and no other constraints, this is the strategy that would evolve. This is the way in which we hope to contribute to the discussion of the evolutionary significance of different transmission strategies.It is not possible to argue positively whether the optimal solution found in our models can be reached, due to evolutionary time constrains or other constraints (which are not taken into account in our simple model). Therefore, the least we can do is provide an argument that relates the synaptic strategy with the infectivity and in vitro growth and consequent plaque formation with vaccinia virus We constructed a mathematical model that describes the dynamics of viral infections, including two transmission pathways: cell-free transmission, and cell-to-cell transmission through viral synapses. This was motivated by the fact that besides free-virus transmission, synaptic spread has recently been suggested to play a major role in HIV infection, with source cells typically transferring tens to hundreds of viruses to the same target cell 2 virus particles are transferred through synapses in HIV infection In the light of this, an explanation is required for the observation that on the order of 10A higher burst size of multiply infected cells could have this effect. While this can indeed elevate the efficiency of passing many viruses per synapse, the increase in burst size must be super-additive for this effect to be observed, e.g. doubly infected cells must produce and transfer more than twice as much virus as singly infected cells. There are currently no data that examine the burst size of infected cells in dependence of the infection multiplicity. A super-additive effect, however, is unlikely to occur unless special cooperative interactions between co-resident viruses occur. Cooperative effects have been observed in the context of unintegrated viral DNA, which could produce offspring virus in the presence of integrated virus rather than becoming a replicative dead end Similarly, while saturation of intracellular defense factors can theoretically make it advantageous to pass many viruses per synapse, the relevance of this mechanism in HIV infection remains unclear. TRIM5α has been identified as an intracellular factor that inhibits HIV replication upon entry into the cell. It has been found to be especially effective at preventing HIV-1 infection in cells derived from Old World monkeys s strategies, a further reduction in the number of transferred viruses does not lead to the infection of a higher number of cells. In this case, “saving” viruses for yet uninfected target cells would be a waste, and it would pay to transfer a larger batch. This argument only holds, however, if the infection probability of free virus lies below a threshold relative to the infection probability during synaptic transmission. Otherwise, the majority of viruses that fail to be transmitted through synapses would likely find target cells via the free virus pathway, and the best synaptic strategy would again be to transfer a single virus per synapse. It is currently unclear whether the life-span of free viruses is sufficiently short relative to that of synaptically transferred viruses. It is feasible that in vivo, the rate of virus loss in the extracellular environment significantly exceeds that occurring during synaptic transmission, even though a sizable amount of virus can be lost in the endocytic pathway during synaptic transmission Constraints during synapse formation can result in an optimal viral strategy where a larger number of viruses is passed per synapse. While it is conceivable that transfer of fewer viruses per synapse leads to the generation of more synapses and the infection of a larger number of cells, there is likely a limit to the number of synapses that can be formed by a source cell during its life-span, because formation of the synapse and the consequent viral transfer take up time, and because each infected cell has only a limited number of neighbors that it can realistically reach by synapse. Thus it is plausible to assume that especially for lower-210 viral particles per synapse does not represent the result of viral adaptation to maximize its fitness. It could be a side-effect of another process the benefit of which outweighs the cost associated with synaptic transmission. The important result in this paper is that in the most basic setting, the transfer of more than one virus per synapse leads to a reduction in the basic reproductive ratio of the virus, and thus to a lower fitness, despite synaptic transmission being very effective for virus transmission on a per cell basis. Hence, more work needs to be performed to account for its prevalent existence in HIV infection.While these are some biological mechanisms that could apply to HIV and render an intermediate number of viruses transferred per synapse advantageous, detailed investigation of the kinetics and measurement of relevant parameters is required to get further insights into whether a given hypothesis can be rejected or whether it is consistent with data. The mechanisms explored here do not take into account some important aspects of the viral evolution in vivo. It is possible that synaptic transmission leads to accelerated evolution of the virus to overcome certain selection pressures, most likely through the multiple infection of cells. Viral recombination is a well-known process that requires multiple infection The work described in this paper is based on ordinary differential equations, which have been explored analytically and numerically. Due to the extensive nature of this analysis, details are given in Supporting Information S1This file provides mathematical details about the modeling approaches described in the main text.(PDF)Click here for additional data file.


Temperature is known to induce changes in mosquito physiology, development, ecology, and in some species, vector competence for arboviruses. Since colonized mosquitoes are reared under laboratory conditions that can be significantly different from their field counterparts, laboratory vector competence experiments may not accurately reflect natural vector-virus interactions.Culex tarsalis strains for West Nile virus (WNV).We evaluated the effects of larval rearing temperature on immature development parameters and vector competence of two Rearing temperature had a significant effect on mosquito developmental parameters, including shorter time to pupation and emergence and smaller female body size as temperature increased. However, infection, dissemination, and transmission rates for WNV at 5, 7, and 14 days post infectious feeding were not consistently affected.Culex tarsalis mosquitoes.These results suggest that varying constant larval rearing temperature does not significantly affect laboratory estimates of vector competence for WNV in  Temperature is a key factor in vector-borne pathogen transmission. It can significantly affect insect behavior and physiology ,2 includCulex tarsalis is a primary vector of West Nile virus (WNV), St Louis encephalitis virus (SLEV), and Western equine encephalitis virus (WEEV) in the western United States [d States -12. Calid States ,14. Thesd States  and lab d States . Colonizd States . TogetheCx. tarsalis development parameters including body size, but did not affect vector competence for WEEV or SLEV [Culex spp. to Rift Valley fever virus (RVFV) or Murray Valley encephalitis virus (MVEV) [Several studies have shown that the influence of extrinsic factors on immature stages extends to the adult stage, possibly affecting susceptibility to pathogens in a species specific manner. Changes in larval water quality affected  or SLEV . Similars (MVEV) ,20. By cs (MVEV) ,21. Othes (MVEV) ,22,23. Cs (MVEV) ,25.Cx. tarsalis demonstrated that decreased larval nutrition impacted several development variables including body size, but did not result in consistent changes in adult vector competence for WNV [Cx. tarsalis[Cx. tarsalis for WNV. We also studied impacts of temperature on various development parameters and body size for Cx. tarsalis.Our previous work with  for WNV . Immatur tarsalis. In addi tarsalis-29. In tCulex tarsalis strains were used for experiments. All mosquitoes were reared at the Wadsworth Center Arbovirus Laboratory. Colonies were originally established from field mosquitoes collected in Coachella Valley, CA (“CV”) and Yolo County, CA (“YC”) in 2003 and 2009, respectively, by WK Reisen. Colony larvae, pupae and adult mosquitoes were reared and maintained under the following controlled conditions: 27°C ± 1°C, 16:8 h light:dark diurnal cycle at approximately 45% relative humidity in 30 × 30 × 30 cm cages. Larvae were reared and fed finely ground koi pellets, ground rabbit pellets and bovine liver powder  at a ratio of 1:1:1. Adults were provided goose blood weekly through a membrane feeder for egg development, and 10% sucrose ad libitum.To control for variation among mosquito genotypes and to account for vector competence changes that possibly occur during the colonization process, two independent Aedes albopictus cells (C636), the titer of the virus stock was 5.0E + 09 PFU/ml. Vero cells were used for blood meal virus titrations and screening mosquito samples for WNV. All experiments with infectious virus were completed in the Wadsworth Center, Arbovirus Laboratory Biosafety Level 3 insectary according to established safety protocols.The WNV strain WN02-1956 (GenBank: AY590222) used for all experiments had been isolated from the kidney of an infected American crow in African green monkey kidney (Vero) cells in 2003. Following one round of amplification in st and 2nd instar larvae, 90 mg for 3rd instar and 180 mg for 4th instar. Preliminary experiments established that this feeding protocol was optimal for larval development [Two replicate experiments were performed each with the CV colony and the YC colony, utilizing three constant temperature treatments at the larval stage . For each temperature, five groups of newly hatched larvae (300 per group) were counted into plastic containers  containing 1 L of filtered water. Larval food was added to each container six days per week in the following amounts: 60 mg for 1elopment .Pupae for each temperature group were pooled in 50 mL conical tubes filled with filtered water and topped with fine mesh and a sugar cube. Pupal maintenance was continued at the experimental temperatures, on a 16:8 h light:dark diurnal cycle with approximately 45% relative humidity. Newly emerged adults were removed to 4.3 L cartons and held at 27°C until blood feeding. The number of pupae, males, and females were recorded six days per week. Adult mosquito wings were measured from the alular notch to the distal margin, excluding the fringe, using Axiovision software and a Zeiss microscope. Time to pupation, time to adulthood, percent pupation, percent emergence and wing length were compared using chi-squared and t-tests.2 anesthesia on a FlyPad  and placed in 4.3 L cardboard containers. Blood fed adults were provided with 10% sucrose ad lib and held for up to 14 days after feeding at 27°C, 16:8 light:dark photoperiod in a growth chamber.Five-to ten-day old female mosquitoes were starved of sugar for four hours prior to blood feeding. Stock WN02-1956 was added to 5 mL defibrinated bovine blood  with 2.5% sucrose solution to average titers of 3.4E + 08 PFU/mL  (Table Effects of rearing temperature on f) Table . In expef) Table .Investigating effects of environmental conditions on mosquito life cycle parameters is important considering that laboratory rearing protocols are usually not representative of the temperature, nutrition, and density fluctuations immature larval stages experience in the field. Generally, mosquitoes collected from the field are significantly smaller than those utilized in laboratory experiments, and studies have shown various other differences, including adult nutritional reserves ,16. SeveCulex tarsalis is a particularly important species because they are found in a variety of habitats and are competent vectors for SLEV, WEEV and WNV [Cx. tarsalis vector competence for WEEV and SLEV have been correlated with changing temperature in Kern County, California, indicating that climate change could facilitate the northward expansion of these viruses [Cx. tarsalis life history parameters, including decreased adult survivorship, adult body size, reproductive effort and immature development time, which was correlated with increasing temperature [ and WNV ,11,12,36 viruses ,29,31. Operature ,37. Addiperature ,23,38,39Cx. tarsalis to become infected, disseminate or transmit WNV. Previously, it has been shown that changes in rearing conditions had no effect on the vector competence of Cx. annulirostris for MVEV, Cx. pipiens for RVFV or Cx. tarsalis for either SLEV or WEEV [Many studies in Aedes mosquitoes have found that altering environmental parameters not only influences immature development, but also vector competence for arboviruses ,22,24,39 or WEEV -20. HoweCulex species vector competence. It remains to be seen whether fluctuating rearing temperature may affect this phenotype [Cx. tarsalis.Our larval studies, in combination with results from other groups, suggest that varying constant larval rearing temperature has no effect on henotype . In addiWNV: West Nile virus; WEEV: Western Equine Encephalitis virus; SLEV: Saint Louis Encephalitis virus; RVFV: Rift Valley Fever virus; MVEV: Murray Valley Encephalitis virus; VEEV: Venezuelan Equine Encephalitis virus; CHIKV: Chikungunya virus; DENV: Dengue virus; SINV: Sindbis virus; LACV: LaCrosse Encephalitis virus; PFU: Plaque forming units; CV: Coachella Valley strain; YC: Yolo County strain; FBS: Fetal bovine serum; MD: Mosquito diluent; dpf: Days post feeding.The authors declare that they have no competing interests.BLD participated in the study design, carried out laboratory experiments, participated in data analysis and participated in drafting the manuscript. LDK participated in the study design and participated in drafting the manuscript. JLR participated in the study design, participated in data analysis and participated in drafting the manuscript. All authors read and approved the final version of the manuscript.


To the Editor: West Nile virus (WNV) circulates mainly in birds and ornithophilic mosquitoes. Humans and horses are considered incidental, dead-end hosts  . The lungs were sent to Onderstepoort Veterinary Institute for African horse sickness RT-PCR. WNV-specific real-time RT-PCR showed positive results. DNA sequencing and phylogenetic analysis identified WNV lineage 2 in several sections of the brain . Results of African horse sickness RT-PCR on lung tissue specimens were inconclusive and could not be confirmed by culture.On May 22, six days after the autopsy on the horse, fever, malaise, myalgia, stiff neck, and severe headache developed in the veterinary student who had handled the horse brain. A rash appeared 2 days later. Symptoms persisted for ≈10 days. The patient was treated symptomatically by an infectious disease specialist and prescribed bed rest. Because cases of Rift Valley fever were recently reported in veterinarians in South Africa, serum was sent to the National Institute for Communicable Diseases, where a virus isolate was obtained in suckling mice and identified as WNV by RT-PCR.After diagnosis of WNV infection in the pony, RNA extracted from the original human serum and from the suckling mouse isolate was sent to the Department of Medical Virology, University of Pretoria, for DNA sequencing and phylogenetic analysis of the virus. Comparison of part of the nonstructural protein 5 gene identified identical sequences from the student’s serum, the virus isolate, and the pony’s brain. All sequences clustered with lineage 2 WNV and were closely related to isolates obtained from horses diagnosed with fatal WNV encephalitis in South Africa in 2008 .,Human infections with WNV have been described after bird autopsies and needle stick injury in laboratory workers (The patient wore latex gloves, his only protection during the autopsy, and had removed the spinal cord and brain. No protective inhalation or eye equipment was worn. No autopsy assistants or other students who worked with or were near the carcass became sick or seroconverted. The most likely route of infection may have involved exposure of mucous membranes to droplets. After the incident, biosafety measures were improved and included wearing of masks and eye protection gear during autopsies at the facility.


C. neoformans and C. gattii are important agents of meningoencephalitis in immunocompromised and immunocompetent hosts, respectively. They are grouped into eight major molecular types, VNI-VNIV for C. neoformans and VGI-VGIV for C. gattii. These major molecular types differ in their host range, epidemiology, antifungal susceptibility and geographic distribution. To enable a rapid identification of the major molecular types and potential hybrids within the two species specific probes based on the PLB1 gene in combination with hyperbranched rolling circle amplification (HRCA) were developed. HRCA was applied to 76 cryptococcal strains, 10 strains each representing the 7 haploid major molecular types, 4 VNIII hybrid strains and 2 inter-species hybrid strains. All strains were correctly identified to the major molecular type and or hybrid type using HRCA alone. To increase the sensitivity a semi-nested PCR step was developed, which will enable the identification of the molecular types/hybrids directly from clinical samples, harboring a low copy number of DNA (40 copies). Thus, HRCA based on the PLB1 locus alone and in combination with a semi-nested PCR showed to be a specific and sensitive methodology, with a great potential to be used on clinical specimens for the direct diagnosis of the agents of cryptococcosis, including hybrid strains, enabling a rapid and patient tailored treatment choice of this disease.The agents of cryptococcosis  Cryptococcus neoformans and C. gattii. Most clinical laboratories do not routinely identify the isolates to species level. However, cryptococcosis is associated with a significant morbidity and mortality and is the most common invasive fungal infection in HIV patients, with an estimated incidence of 1 million cases annually C. neoformans. C. gattii on the other hand affects mostly immunocompetent individuals C. neoformans molecular types correlate with the serotypes: VNI/AFLP1, serotype A; VNII/AFLP1A, serotype A; VNIII/AFLP3, serotype AD; and VNIV/AFLP2, serotype D. The molecular types of C. gattii  are all associated with both serotypes B and C Cryptococcosis is a systemic mycosis acquired by inhalation of infectious propagules (desiccated yeasts cells or spores) produced by the basidiomycetous yeasts C. neoformans and C. gattii differ in their epidemiological C. gattii often have a worse prognosis than those caused by C. neoformansThe major molecular types of Cryptococcus species are identified after culturing the isolates from a clinical sample followed by biochemical tests, which may delay the final diagnoses C. neoformans and C. gattii are then determined using a variety of molecular typing techniques The currently used laboratory identification model for the agents of cryptococcosis has major limitations. Molecular methods have the advantage of being highly sensitive and specific to overcome the limitations of conventional diagnosis C. neoformans and C. gattii” developed a consensus MultiLocus Sequence Typing (MLST) scheme for the members of the C. neoformans/ C. gattii species complex based on the variable regions within the capsular associated protein gene (CAP59), glyceraldehyde-3-phosphate dehydrogenase gene (GPD1), laccase (LAC1), phospholipase (PLB1), Cu, Zn superoxide dismutase (SOD1), orotidine monophosphate pyrophosphorylase (URA5) genes and intergenic spacer region (IGS1), to standardize, increase the discriminatory power and improve the inter-laboratory reproducibility of cryptococcal genotyping http://mlst.mycologylab.org.In 2009, the ISHAM working group on “Genotyping C. neoformans and C. gattii and to differentiate between the serotypes A and D of C. neoformansThe recently developed technique of hyperbranched rolling circle amplification (HRCA) C. neoformans/ C. gattii species complex is still lacking. The current study aimed to develop specific probes in combination with HRCA to identify the eight major molecular types (VNI-VNIV and VGI-VGIV) within this species complex. This methodology should allow for a fast identification of the agents, the major molecular types and hybrids involved in human and animal cryptococcosis from DNA extracts from pure cultures or directly from various clinical specimens, which will enable an informed choice of early antifungal therapy, and provide a highly sensitive tool for epidemiological surveillance.Besides a number of available molecular identification and typing techniques, a fast, easy and highly standardized method for the identification of the major molecular types and potential hybrids within the C. neoformans/ C. gattii species complex, maintained at the Westmead Hospital Culture Collection, Sydney, Australia, were studied , C. dubliniensis (WM 602 = CBS 7987 T), C. krusei (WM 14 = CBS 573 T), C. globosa (WM 284 = CBS 599 T), Scedosporium prolificans (WM 06.502), Aspergillus fumigatus (WM 06.262), Fusarium solani (WM 07.291) strains were also studied. All the strains had been previously identified by routine biochemical methods and ITS sequencing and are maintained at the Westmead Hospital Culture Collection, Sydney, Australia and/or the CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands.Ten isolates of each of the seven haploid major molecular types of the  studied . These s studied , amountiet al.CAP59, GPD1, LAC1, SOD1, URA5, PLB1 and IGS, were amplified according to the consensus MLST scheme for C. neoformans and C. gattiiGPD1 and LAC1 of C. neoformans. The GPD1 locus of C. neoformans was amplified using the primers GPD1cn-F 5′ATGGTCGTCAAGGTTGGAAT 3′ and GPD1cn-R 5′ GTATTCGGCACCAGCCTCA 3′, and the LAC1 locus of C. neoformans was amplified using the primers LAC1cn-F 5′ GGCGATACTATTATCGTA3′ and LAC1cn-R 5′-TTCTGGAGTGGCTAGAGC3′ . Allele (AT) and sequence types (ST) were assigned according to the MLST database at http://mlst.mycologylab.org and new AT’s and ST’s were added to this database.DNA extractions were performed according to Ferrer PLB1 gene was performed in a final volume of 50 µL. Each reaction contained 50 ng of DNA, 1X PCR buffer (200 mM Tris-HCl (pH 8.4), 500 mM KCl - Invitrogen), 0.2 mM each of dATP, dCTP, dGTP, and dTTP (Invitrogen), 2 mM magnesium cloride, 1.5 U Taq DNA polymerase (Invitrogen), and 50 ng of each primer PLB1-F (5′ CTTCAGGCGGAGAGAGGTTT 3′) and PLB1-R (5′ GATTTGGCGTTG GTTTCAGT 3′) Amplification of the PLB1 gene of C. neoformans and C. gattii were designed (PLB1 gene amplification (see above), using 15 µl of the initial amplicon.To enhance the sensitivity of the target DNA detection a semi-nested PCR strategy was developed. Nested-primers with 100% homology to the designed  using sedesigned . The conC. neoformans  C. gattii . The selection of informative SNPs was based on sequences maintained in the C. neoformans/ C. gattii MLST database from the Molecular Mycology Laboratory, Sydney University, Australia, http://mlst.mycologylab.org, and additional sequences generated as part of the current study. The sequences used for the SNP analysis included seven unlinked genetic loci: the housekeeping genes CAP59, GPD1, LAC1, PLB1, SOD1, URA5 and the IGS1 region. They originated from 232 C. neoformans  and 359 C. gattii  global strains. To identify specific SNPs for each of the seven major haploid molecular types the sequences from the 591 C. neoformans and C. gattii isolates were aligned using the program MEGA version 5 Padlock probes were designed to target specific single nucleotide polymorphism (SNP) for each of the seven major haploid molecular types of the et al.In addition, the primers RCA1 and RCA2 , which aPLB1 gene either by single or semi-nested PCR (see above), the amplicons were purified with the PureLink PCR purification kit . The ligation of the padlock probes to the amplified PCR products was performed according to Wang et al.2, 0.1% Igepal, 0.01 mMrATP, 1 mMDTT. The ligation reaction conditions included 5 min denaturation at 94°C followed by 15 cycles of 94°C for 30 s and 4 min ligation at 65°C. The ligation mixture was then subjected to exonucleolysis to remove non-circularized padlock probe and excess PCR product in order to reduce subsequent ligation-independent amplification events. The exonuclease digestion was performed in a volume of 20µl by adding 10 U of each exonucleases I and III  to the ligation solution and incubating it at 37°C for 30 min followed by 94°C for 30 s to inactivate the exonuclease.After amplification of the Bst DNA polymerase (New England Biolabs), 400 µM deoxynucleoside triphosphate mix, 10 pmol of each RCA primers . In addition BLAST searches using the primer sequences reveal only a homology to either C. neoformans or C. gattii and did not find any matches to other basidiomycetious yeasts such as C. laurentii, C. albidus, C. uniguttulatus or Trichosporon spp.To demonstrate the specificity of the used primers, the C. neoformans VNIII hybrids  studied generated positive signals with the respective HRCA probes , which resulted in the same fragments as the ones obtained for the major molecular type VGIV.HRCA amplification in combination with the detection of the generated products on a real-time-PCR was applied to ten representative strains of each major haploid molecular type , as well hybrids  and 2 in hybrids . A signaA probes . The proPLB1 locus was compared against the GenBank database it showed BLASTN hits with greater than 98% identity to the C. neoformans and C. gattii PLB1 gene only. This demonstrated that it has a high specificity for C. neoformans and C. gattii. As such this primer was used in combination with the PLB1-R primer PLB1 amplification as a template to amplify a fragment of 607 bp , which is essential for the direct detection of the molecular types or species from clinical specimens  and C. gattii (VGI-VGIV) are not identified in routine laboratories and the virulence of the different genotypes has not been systematically studied, although those molecular types have molecular, epidemiological, serological and antifungal susceptibility differences C. neoformans were associated with a higher mortality among HIV patients in sub-Saharan Africa C. neoformans hybrids strains were associated with increased mortality in humans, although they had attenuated virulence in mouse models Improved technologies, which accurately identify the different molecular types of CAP59C. neoformans and C. gattii, but was not able to differentiate the serotypes B and C of C. gattii. Kaocharoen et al.C. neoformans and C. gattii using the internal transcribed spacer (ITS) regions of the rDNA gene cluster as a target. However, this enabled only the differentiation between VNI/VNII, VNIV and C. gattii. Feng et al. developed a duplex PCR assay using vacuolar membrane gene to differentiate between the molecular types of C. gattiiC. neoformans. The current study detected specific SNPs for each of the major haploid molecular types of C. neoformans (VNI-VNIV) and C. gattii (VGI-VGIV) in the PLB1 locus, allowing for the development of specific padlock probes. HRCA using the PLB1 locus was able to differentiate the seven major haploid molecular types of C. neoformans  and C. gattii  and also identified correctly different types of hybrid isolates from DNA extracts from pure cultures.Specific padlock probes in combination with hyperbranched rolling circle amplification (HRCA) are ideal for the development of diagnostic assays, which require speed, specificity and reproducibility. The application of loop-mediated isothermal DNA amplification (LAMP) using the capsule-associated gene C. neoformansTo enable a direct detection form clinical specimens the sensitivity was increased via the development of a semi-nested PCR. This increased the detection limit to approximately 1 pg of DNA equaling a minimum of 40 copies of DNA. This brings the detection limit in the range of the DNA amounts being detected by multicopy regions, such as the ribosomal gene cluster, which is widely used as a target for the detection of fungal disease agents, where the lower detection limits range from 10 fg PLB1 locus alone or together with the application of the herein developed semi-nested PCR approach showed to be a specific and highly sensitive methodology, with a great potential to be used on DNA extracts from pure cultures or clinical specimens for the direct identification of the different major molecular types and potential hybrids of the agents of human and animal cryptococcosis, providing the basis for a rapid and patient tailored treatment choice for this disease.For the first time HRCA of the Table S1Accession number of the sequences included in the study to design the semi-nested PCR primer.(XLS)Click here for additional data file.


Human activity is causing new encounters between viruses and plants.Anthropogenic interventions include changing land use, decreasing biodiversity,trade, the introduction of new plant and vector species to native landscapes,and changing atmospheric and climatic conditions. The discovery of thousands ofnew viruses, especially those associated with healthy-appearing native plants,is shifting the paradigm for their role within the ecosystem from foe to friend.The cost of new plant virus incursions can be high and result in the loss oftrade and/or production for short or extended periods. We present and justifythree recommendations for plant biosecurity to improve communication about plantviruses, assist with the identification of viruses and their impacts, andprotect the high economic, social, environmental, and cultural value of ourrespective nations' unique flora: 1) As part of the burden of proof,countries and jurisdictions should identify what pests already exist in, andwhich pests pose a risk to, their native flora; 2) Plant virus sequences notassociated with a recognized virus infection are designated as “unculturedvirus” and tentatively named using the host plant species of greatestknown prevalence, the word “virus,” a general location identifier,and a serial number; and 3) Invest in basic research to determine the ecology ofknown and new viruses with existing and potential new plant hosts and vectorsand develop host-virus pathogenicity prediction tools. These recommendationshave implications for researchers, risk analysts, biosecurity authorities, andpolicy makers at both a national and an international level. Over the last 60 years, global trade has changed significantly with the formation ofthe World Trade Organization (WTO) in 1995 A pest is “any species, strain or biotype of plant, animal or pathogenic agentinjurious to plants or plant products,” as defined in a glossary ofphytosanitary terms by the International Standards for Phytosanitary Measures (ISPM)may be associated with the importedmaterial. Therefore, a shared burden of proof exists between trading countries foridentifying the biosecurity risk of an organism.At least three parties are involved in international trading transactions: exporters,biosecurity agencies, and importers, but the responsibility for identifying whethernew viruses are: 1) present in a country or region and 2) associated with a risk tothe local flora is not always clear. It is common for the importing country to adopta conservative stance and limit the importation of plant material based on the riskof a pathogen that A major implication of plant virus discovery is the impact of new viruses on plantbiosecurity. There are at least three distinct motivations for plant virusdiscovery: 1) identifying causes of viral diseases in crops, 2) screening forspecific viruses when their presence is suspected, and 3) sequencing of virusesregardless of symptoms in the target plant or botanical ecosystem. The third andmost recent motivation is dependent on the development and implementation of newsequencing technologies resulting in the discovery of new species of viruses andother microorganisms Using deep sequencing technologies has allowed the identification of known and newviruses and provided a new understanding of their biodiversity. In addition to thetraditional “pathogenic” role of plant viruses, more intimate symbioticrelationships such as (a) antagonism (one partner benefits at the expense of theother and can manifest in a disease), (b) commensalism (one partner benefits whilethe other is unaffected), or (c) mutualism (both partners benefit) have beenproposed We present and justify three recommendations for plant virus characterization andclassification in the context of new virus discovery. These will permit bettercommunication about plant viruses, assist with the identification of viruses andtheir impacts, and protect the high economic, social, environmental, and culturalvalue of our respective nations' unique flora.As the knowledge of virus identity is extended, an equal, if not increased, effort isrequired to discover viruses' roles in their existing and potential newecosystems. In particular, since many newly discovered viruses will be derived frominfections of native plant hosts, it is vitally important for biosecurity agenciesto survey for the presence of plant viruses in their own native flora.Potyviridae were identified only occasionally inthe TGP  by contrast to more than 10% of theinfected plants in the ACG Totiviridae, Partitiviridae, Chrysoviridae,and Endornaviridae were prevalent in both studies, accounting forabout half of the viruses found. Several detected virus sequences proved difficultto classify in specific genera, having properties suggesting their membership inmultiple genera or in a novel genus within a family Searches for virus-related sequences in ecogenic and metagenomic discovery projectsof natural ecosystems have located many putative viruses. The Lake Needwood projectresulted in identification of sequences related to 65 known plant viruses At the policy level in many countries, risk analyses are applied as decision-makingtools that include the identification and assessment of the risk or hazard, theconsequences of establishment, and appropriate risk management strategies.Decision-making tools to support pest risk analyses require easy and rapid access toreliable sources of information relating to the plant commodities and native floraat risk. The WTO mandate to demonstrate freedom of a pest as being “known notto occur” within a country or region forms the basis of biosecurity policy forthe movement of plant material around the globe. This burden of proof drives theidentification of existing and pest plant virus species within a country, includingwithin native flora. For instance, the New Zealand Biosecurity Act For the majority of these virus sequences, it is unclear whether they represent anintact virus (rather than a remnant of a virus expressed from the plant genome) (seeRecommendation 2), or whether it has an antagonistic, commensal, and/or amutualistic relationship with the plant host. More importantly, it is unclearwhether the sequenced virus has the capacity to alter that relationship in a newplant host or environment, induce disease symptoms in a new host plant, or recombinewith endemic viruses to result in a more virulent or new pathogenic virus species(Recommendation 3).The distinction between a virus as a physical entity and an in-silico virus speciesthat encompasses actual viruses is important when considering naming of virusesth contig of the 101st plant sampled in 2005 on the TGPPreserve). Phylogenetic analysis of the retrieved viruslike sequences identifies themost closely related known viral sequences that have been lodged on public databasesand determines if the obtained viral sequences are representative of a known orpotentially new viral species.Naming putative viruses is initially for laboratory convenience. For example, in theTGP study, names comprised an identifying number of the source plant plus the numberof the sequence contig generated by sequence assembly, as in 05TGP00101.25 , and thelocation of first isolation (see criterion 3).The names must be unique and nonambiguous. It is possible fordistinct viral species to have the same host species of original isolationand cause the same disease symptoms. Solutions to naming ambiguity problemshave already been implemented by the ICTV. A geographical qualifier can beplaced before the “virus” in the name, for example inHibiscus latent Singapore virus (HLSV). Marinebacteriophages are identified by names specifying host, followed by φand a non-word identifier, such as in Prochlorococcusmarinus φ P-SSM2 Grapevine leafroll–associated virus 3,has been adopted.The names should not be based on the symptoms caused by infection withthe virus. Recent discoveries of viruses in healthy-appearingplants reveal the folly of using disease symptoms in the name. Not only aremany of the new viruses not associated with symptoms in their host ofisolation, but the same virus also can cause different symptoms on differentplants or plant varieties.Taxonomic indications should be avoided. Adding a generictaxonomic prefix to “virus,” for example in“Tobacco mosaic tobamovirus” has beenpresented and discussed The naming convention should allow some means of identifying that thevirus is a “sequence-only” virus. The consensus viralgenome sequences that result from in-silico assembly and analysis of deepsequencing datasets are not considered acceptable for GenBank/EMBL/DDBJdeposition and are labeled as coming from an “uncultured virus.”It is reasonable therefore that viruses recognized only by their sequencesignatures have a lower taxonomic status than fully characterized virusesand adding a “p-,” “e-,” or “s-” prefix,respectively for putative, electronic, or sequence, before the word virus ispossible.An initial biosecurity investigation might escalate to a full investigation orincursion response when a new or regulated virus, not yet established, is detectedwithin the country's environment. Risk assessment and analysis are the firststeps in assessing the economic, social, environmental, or cultural impacts of thenew incursion. If the impacts are high, quarantine agencies will assess the variousoptions available. If the likely impacts are low, normally, no further officialaction is taken. It is therefore important to understand the existing and potentialplant virome in our ecosystems. Viruses are part of the wider ecosystem and someinteractions may result in disease while other interactions may be beneficial to thehost plant.Cassava mosaic virus that haveresulted in many farmers abandoning cassava cultivation and consequentlydestabilizing food security in east Africa Bemisia tabaci, into Brazil in the early 1990s resultedin the horizontal transfer of previously unrecorded indigenous begomoviruses fromnative onto cultivated plants Some of the more serious plant virus disease epidemics are the result of theintroduction of either the host plant or the insect vector into a new region thatexposes the crop plant to an endemic or “native” virus, resulting indisease. For example, introduction of cassava into sub-Saharan Africa has seen theemergence of aggressive strains of Plant viruses range between specialists, with only a single plant host, togeneralists that have many hosts. For virus infection to occur, the plant host willlack the defense mechanisms to protect against infection of the invading virus www.promedmail.org). Theseincursions often result in trade restrictions and the introduction of strictphytosanitary measures on the local industry to prevent further spread and/or toeradicate the pest.The intercontinental mobilization of the human population has assisted the spread ofinfectious diseases on a global scale. This was clearly demonstrated for over halfthe Australian potyvirus species that have been introduced during the last twocenturies by European migrants The deep sequencing strategy currently used for plant virus discovery posessignificant challenges for biosecurity agencies, particularly with respect to thelarge number of new plant virus sequences that are being discovered and have thepotential to cause disease. Concurrently, there is an increasing number of reportsof plant viruses as mutualists, which can provide benefits to the host plant In light of these challenges, we have proposed three recommendations to be consideredby biosecurity agencies and have identified research gaps that need to befilled.Globally, communities are demanding improved conservation strategies for native floraand fauna, as demonstrated in the New Zealand Biosecurity Act Currently, plant quarantine is based on the presence/absence of the pest/pathogen andnot the presence of disease that it may cause. As such, in-silico viruses that arebeing discovered using deep sequencing currently have the potential to disrupt themovement of plant material based on the perception that the virus sequence may causea disease in a new environment. The second recommendation of this paper is thatplant virus sequences that have not been associated with virus infection in a hostplant are designated as “uncultured virus” to indicate that theassembled genome sequence is “sequence-only.”The biological and ecological function of “sequence-only” viruses thatare discovered in plants using sequencing strategies is largely unknown The cost of plant virus incursions is high and can result in the loss of trade and/orproduction for extended periods. The three recommendations for plant biosecuritywill permit better communication about plant viruses, assist with the identificationof viruses and their impacts, facilitate the development of sound biosecuritypolicy, and protect the high economic, social, environmental, and cultural value ofunique flora.


In China in 2010, a disease outbreak in egg-laying ducks was associated with a flavivirus. The virus was isolated and partially sequenced. The isolate exhibited 87%–91% identity with strains of Tembusu virus, a mosquito-borne flavivirus of the Ntaya virus group. These findings demonstrate emergence of Tembusu virus in ducks. From June through November 2010 in the People’s Republic of China, a disease characterized by a sudden onset was observed on many egg-laying and breeder duck farms. Egg production in affected ducks dropped severely within 1–2 weeks after disease onset. Other consistent signs included acute anorexia, antisocial behavior, rhinorrhea, diarrhea, ataxia, and paralysis. Rate of illness was usually high (up to 90%), and mortality rates varied from 5% to 30%. From affected ducks we isolated and identified a Tembusu virus (TMUV).During the outbreak, we examined 11 diseased ducks  from 5 duck farms in 4 provinces. At necropsy, viscera samples  were collected and placed in 10% buffered formalin. Sections were embedded in paraffin and stained with hematoxylin and eosin. The theca folliculi from each duck were also used for virus isolation or detection by PCR.The main pathologic changes observed consistently in almost all diseased ducks were found in the ovaries: hyperemia, hemorrhage, degeneration, distortion, macrophage and lymphocyte infiltration, and hyperplasia; in the liver, interstitial inflammation was found in the portal area  search in GenBank. Unexpectedly, a 221-nt sequence (GenBank accession no. HQ641388) was shown to encode the nonstructural (NS) 1 protein of flavivirus, which exhibited 73%–85% identity to flaviviruses in the Ntaya virus and Japanese encephalitis virus groups, such as Bagaza virus (GenBank accession no. ACG60714 [4]). The result demonstrated the possible presence of a flavivirus in ducks.Because eastern equine encephalitis (EEE) virus has been shown to cause paralysis in Pekin ducks   of part of the NS3 genomic region obtained from the isolate was 66%–77% identical to the corresponding sequence of viruses in the Ntaya virus and Japanese encephalitis virus groups. The amplicon sequences from the 11 theca folliculus samples shared 98%–100% identity with the YY5 isolate. PCR testing of another 52 samples from diseased ducks from the 4 provinces detected flavivirus-specific RNA in 29 samples. Overall, 40 (63.5%) of 63 samples were positive for flavivirus. Flavivirus-specific RNA was most frequently detected in theca folliculi, followed by intestinal mucosa, uterus, spleen, trachea, cloaca (swab), and liver .Subsequently, the YY5 isolate was injected intramuscularly into nine 55-week-old Pekin ducks and ten 30-week-old Shaoxing ducks. At day 4 postinoculation, the pathologic changes were reproduced in these experimentally infected ducks. The flavivirus RNA was detected by NS3-based RT-PCR, and the virus was again isolated from theca folliculi.To investigate the genetic relationship of the isolate with flaviviruses, we obtained the genomic sequence of a 1,035-bp segment at the 3′ terminus of the NS5 gene (GenBank accession no. HQ641390) from strain YY5 by RT-PCR and primers FU1 and cFD3 as described . ComparaFlavivirus  of 15 theca folliculus samples, suggesting that reproductive tissues may be a major site for viral persistence, replication, or both. Experimental infections further confirmed that TMUV can be reisolated from theca folliculi. These results suggested that TMUV may be the causative agent of duck hemorrhagic ovaritis.Because TMUV belongs to the mosquito-borne virus cluster of flaviviruses, mosquitoes might be involved in the spread of this virus. Detection of the virus in cloacal swab samples suggests probable horizontal transmission through ingestion or inhalation of feces-contaminated material.Culex, but the disease associated with TMUV infection was not known. However, a chick-origin TMUV isolate, originally named Sitiawan virus, can cause encephalitis and retarded growth in broiler chicks (TMUV was originally isolated from mosquitoes of the genus


It is generally thought that viruses require the cytoskeleton during their replication cycle. However, recent experiments in our laboratory with rubella virus, a member of the family Togaviridae (genus rubivirus), revealed that replication proceeded in the presence of drugs that inhibit microtubules. This study was done to expand on this observation.The replication of three diverse viruses, Sindbis virus , vesicular stomatitis virus , and Herpes simplex virus (family Herpesviridae), was quantified by the titer  produced in cells treated with one of three anti-microtubule drugs  or the anti-actin filament drug, cytochalasin D. None of these drugs affected the replication these viruses. Specific steps in the SINV infection cycle were examined during drug treatment to determine if alterations in specific steps in the virus replication cycle in the absence of a functional cytoskeletal system could be detected, i.e. redistribution of viral proteins and replication complexes or increases/decreases in their abundance. These investigations revealed that the observable impacts were a colchicine-mediated fragmentation of the Golgi apparatus and concomitant intracellular redistribution of the virion structural proteins, along with a reduction in viral genome and sub-genome RNA levels, but not double-stranded RNA or protein levels.The failure of poisons affecting the cytoskeleton to inhibit the replication of a diverse set of viruses strongly suggests that viruses do not require a functional cytoskeletal system for replication, either because they do not utilize it or are able to utilize alternate pathways when it is not available. It is generally thought that viruses require the cytoskeleton during infection [There are three major components to the cytoskeleton; actin filaments , intermenfection , althougnfection . To see Given our finding that three diverse viruses replicate to similar titers in the absence or presence of anti-cytoskeletal drugs, we hypothesized that either these viruses do not need the cytoskeletal system or use alternate pathways when it is not available. Since these hypotheses could apply differentially to the steps in the virus replication cycle, we used SINV to investigate whether the anti-cytoskeleton drugs had effects on specific steps in the virus replication cycle. SINV produces four nonstructural proteins (nsP1-4) that are involved in RNA-dependent RNA synthesis occurring in membranous structures in the cytoplasm of infected cells -8. UsingSince colchicine has previously been shown to disrupt the Golgi , wheat gIn summary, following studies in our lab with rubella virus  which foThe authors declare that they have no competing interest.JDM carried out the research and drafted the manuscript. RM and CS also carried out the research for their MS degrees. TKF, as senior author, advised JDM, RM and CS on the research, participated in drafting of the manuscript, and serves as corresponding author. All four authors have read and approved the final manuscript.RM is attending vet school at the University of Georgia. JDM is a Postdoctoral Fellow in the Department of Pathology, Emory University School of Medicine.