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In JoVE (1)

Other Publications (43)

Articles by Slobodan Paessler in JoVE

 JoVE Immunology and Infection

In Vivo Imaging Systems (IVIS) Detection of a Neuro-Invasive Encephalitic Virus

1Experimental Pathology, University of Texas Medical Branch


JoVE 4429

Utilizing luciferase and in vivo imaging systems (IVIS) as a novel means to identify disease endpoints before clinical developments occur. IVIS has allowed us to visualize in real time the invasion of encephalitic viruses over multiple days, providing a more accurate disease model for future study. It has also allowed us to identify the potential protective features of antivirals and vaccines faster than currently utilized animal models. The capability to utilize individual animals over multiple time points ensures reduced animal requirements, costs, and overall morbidity to the animals utilized ensuring a more humane and more scientific means of disease study.

Other articles by Slobodan Paessler on PubMed

Recombinant Sindbis/Venezuelan Equine Encephalitis Virus is Highly Attenuated and Immunogenic

Venezuelan equine encephalitis virus (VEEV) is an important, naturally emerging zoonotic virus. VEEV was a significant human and equine pathogen for much of the past century, and recent outbreaks in Venezuela and Colombia (1995), with about 100,000 human cases, indicate that this virus still poses a serious public health threat. The live attenuated TC-83 vaccine strain of VEEV was developed in the 1960s using a traditional approach of serial passaging in tissue culture of the virulent Trinidad donkey (TrD) strain. This vaccine presents several problems, including adverse, sometimes severe reactions in many human vaccinees. The TC-83 strain also retains residual murine virulence and is lethal for suckling mice after intracerebral (i.c.) or subcutaneous (s.c.) inoculation. To overcome these negative effects, we developed a recombinant, chimeric Sindbis/VEE virus (SIN-83) that is more highly attenuated. The genome of this virus encoded the replicative enzymes and the cis-acting RNA elements derived from Sindbis virus (SINV), one of the least human-pathogenic alphaviruses. The structural proteins were derived from VEEV TC-83. The SIN-83 virus, which contained an additional adaptive mutation in the nsP2 gene, replicated efficiently in common cell lines and did not cause detectable disease in adult or suckling mice after either i.c. or s.c. inoculation. However, SIN-83-vaccinated mice were efficiently protected against challenge with pathogenic strains of VEEV. Our findings suggest that the use of the SINV genome as a vector for expression of structural proteins derived from more pathogenic, encephalitic alphaviruses is a promising strategy for alphavirus vaccine development.

Generation and Characterization of Closely Related Epizootic and Enzootic Infectious CDNA Clones for Studying Interferon Sensitivity and Emergence Mechanisms of Venezuelan Equine Encephalitis Virus

Venezuelan equine encephalitis virus (VEEV) is a reemerging pathogen and a continuing threat to humans and equines in the Americas. Identification of the genetic determinants that enable epizootic VEEV strains to arise and exploit equines as amplification hosts to cause widespread human disease is pivotal to understanding VEE emergence. The sensitivity to murine alpha/beta interferon-mediated antiviral activity was previously correlated to the epizootic phenotype of several VEEV strains. Infectious cDNA clones were generated from an epizootic subtype IC VEEV strain (SH3) isolated during the 1992 Venezuelan outbreak and a closely related enzootic, sympatric subtype ID strain (ZPC738). These VEEV strains had low-cell-culture-passage histories and differed by only 12 amino acids in the nonstructural and structural proteins. Rescued viruses showed similar growth kinetics to their parent viruses in several cell lines, and murine infections resulted in comparable viremia and disease. Unlike what was found in other studies of epizootic and enzootic VEEV strains, the sensitivities to murine alpha/beta interferon did not differ appreciably between these epizootic versus enzootic strains, calling into question the reliability of interferon sensitivity as a marker of epizootic potential.

The Hamster As an Animal Model for Eastern Equine Encephalitis--and Its Use in Studies of Virus Entrance into the Brain

Eastern equine encephalitis virus (EEEV) produces the most severe human arboviral diseases in the United States, with mortality rates of 30%-70%. Vasculitis associated with microhemorrhages in the brain dominates the pathological picture in fatal human eastern equine encephalitis, and neuronal cell death is detectable during the late stage of the disease. We describe use of the golden hamster to study EEEV-induced acute vasculitis and encephalitis. In hamsters, EEEV replicates in visceral organs, produces viremia, and penetrates the brain. The pathological manifestations and antigen distribution in the brain of a hamster are similar to those described in human cases of EEEV.

Experimental Everglades Virus Infection of Cotton Rats (Sigmodon Hispidus)

Everglades virus (EVEV), an alphavirus in the Venezuelan equine encephalitis (VEE) serocomplex, circulates among rodents and vector mosquitoes and infects humans, causing a febrile disease sometimes accompanied by neurologic manifestations. EVEV circulates near metropolitan Miami, which indicates the potential for substantial human disease, should outbreaks arise. We characterized EVEV infection of cotton rats in South Florida, USA, to validate their role in enzootic transmission. To evaluate whether the viremia induced in cotton rat populations regulates EVEV distribution, we also infected rats from a non-EVEV-endemic area. Viremia levels developed in rats from both localities that exceeded the threshold for infection of the vector. Most animals survived infection with no signs of illness, despite virus invasion of the brain and the development of mild encephalitis. Understanding the mechanisms by which EVEV-infected cotton rats resist clinical disease may be useful in developing VEE therapeutics for equines and humans.

Venezuelan Equine Encephalitis Virus in the Guinea Pig Model: Evidence for Epizootic Virulence Determinants Outside the E2 Envelope Glycoprotein Gene

Epizootic strains of Venezuelan equine encephalitis virus (VEEV) cause epidemics by exploiting equines as highly efficient amplification hosts for mosquito transmission. Although phylogenetic studies indicate that epizootic VEEV strains emerge via mutation from enzootic progenitors that are incapable of efficient equine amplification, the molecular mechanism(s) involved remain enigmatic. The convergent evolution of E2 envelope glycoprotein mutations suggests that they are critical to VEEV emergence, but little is known about the role of non-envelope genes. We used the guinea pig, the small animal model that best predicts the ability to generate equine viremia, to assess the role of envelope versus other mutations in the epizootic phenotype. Using reciprocal chimeric viruses generated by swapping the envelope genes of closely related epizootic IC and enzootic ID strains, infections of guinea pigs demonstrated that envelope and non-envelope genes and sequences both contributed to virulence. However, early replication in lymphoid tissues appeared to be primarily envelope dependent.

Venezuelan Equine Encephalitis Virus Infection of Spiny Rats

Enzootic strains of Venezuelan equine encephalitis virus (VEEV) circulate in forested habitats of Mexico, Central, and South America, and spiny rats (Proechimys spp.) are believed to be the principal reservoir hosts in several foci. To better understand the host-pathogen interactions and resistance to disease characteristic of many reservoir hosts, we performed experimental infections of F1 progeny from Proechimys chrysaeolus collected at a Colombian enzootic VEEV focus using sympatric and allopatric virus strains. All animals became viremic with a mean peak titer of 3.3 log10 PFU/mL, and all seroconverted with antibody titers from 1:20 to 1:640, which persisted up to 15 months. No signs of disease were observed, including after intracerebral injections. The lack of detectable disease and limited histopathologic lesions in these animals contrast dramatically with the severe disease and histopathologic findings observed in other laboratory rodents and humans, and support their role as reservoir hosts with a long-term coevolutionary relationship to VEEV.

Noncytopathic Replication of Venezuelan Equine Encephalitis Virus and Eastern Equine Encephalitis Virus Replicons in Mammalian Cells

Venezuelan equine encephalitis (VEE) and eastern equine encephalitis (EEE) viruses are important, naturally emerging zoonotic viruses. They are significant human and equine pathogens which still pose a serious public health threat. Both VEE and EEE cause chronic infection in mosquitoes and persistent or chronic infection in mosquito-derived cell lines. In contrast, vertebrate hosts infected with either virus develop an acute infection with high-titer viremia and encephalitis, followed by host death or virus clearance by the immune system. Accordingly, EEE and VEE infection in vertebrate cell lines is highly cytopathic. To further understand the pathogenesis of alphaviruses on molecular and cellular levels, we designed EEE- and VEE-based replicons and investigated their replication and their ability to generate cytopathic effect (CPE) and to interfere with other viral infections. VEE and EEE replicons appeared to be less cytopathic than Sindbis virus-based constructs that we designed in our previous research and readily established persistent replication in BHK-21 cells. VEE replicons required additional mutations in the 5' untranslated region and nsP2 or nsP3 genes to further reduce cytopathicity and to become capable of persisting in cells with no defects in alpha/beta interferon production or signaling. The results indicated that alphaviruses strongly differ in virus-host cell interactions, and the ability to cause CPE in tissue culture does not necessarily correlate with pathogenesis and strongly depends on the sequence of viral nonstructural proteins.

A Novel, Rapid Assay for Detection and Differentiation of Serotype-specific Antibodies to Venezuelan Equine Encephalitis Complex Alphaviruses

An epitope-blocking enzyme-linked immunosorbent assay was developed for the rapid differentiation of serologic responses to enzootic variety IE and ID versus epizootic variety IAB and IC strains of Venezuelan equine encephalitis (VEE) virus. Two monoclonal antibodies that differentially recognize epizootic versus enzootic VEE virus epitopes were used to measure the serotype-specific blocking abilities of antibodies in sera of naturally infected humans, equines, and bovines, as well as in experimentally infected equines. The assay is simple, species-independent, rapid, and sensitive, and will improve surveillance for VEE emergence. It could also be used to determine the epidemic potential of a VEE virus following an intentional introduction for bioterrorism.

Envelope Glycoprotein Mutations Mediate Equine Amplification and Virulence of Epizootic Venezuelan Equine Encephalitis Virus

Epidemics of Venezuelan equine encephalitis (VEE) result from high-titer equine viremia of IAB and IC subtype viruses that mediate increased mosquito transmission and spillover to humans. Previous genetic studies suggest that mutations in the E2 envelope glycoprotein allow relatively viremia-incompetent, enzootic subtype ID strains to adapt for equine replication, leading to VEE emergence. To test this hypothesis directly, chimeric VEEV strains containing the genetic backbone of enzootic subtype ID strains and the partial envelope glycoprotein genes of epizootic subtype IC and IAB strains, as well as reciprocal chimeras, were used for experimental infections of horses. Insertion of envelope genes from two different, closely related enzootic subtype ID strains into the epizootic backbones resulted in attenuation, demonstrating that the epizootic envelope genes are necessary for the equine-virulent and viremia-competent phenotypes. The partial epizootic envelope genes introduced into an enzootic ID backbone were sufficient to generate the virulent, viremia-competent equine phenotype. These results indicate that a small number of envelope gene mutations can generate an equine amplification-competent, epizootic VEEV from an enzootic progenitor and underscore the limitations of small animal models for evaluating and predicting the epizootic phenotype.

Variation in Interferon Sensitivity and Induction Among Strains of Eastern Equine Encephalitis Virus

Eastern equine encephalitis virus (EEEV) causes human encephalitis in North America (NA), but in South America (SA) it has rarely been associated with human disease, suggesting that SA strains are less virulent. To evaluate the hypothesis that this virulence difference is due to a greater ability of NA strains to evade innate immunity, we compared replication of NA and SA strains in Vero cells pretreated with interferon (IFN). Human IFN-alpha, -beta, and -gamma generally exhibited less effect on replication of NA than SA strains, supporting this hypothesis. In the murine model, no consistent difference in IFN induction was observed between NA and SA strains. After infection with most EEEV strains, higher viremia levels and shorter survival times were observed in mice deficient in IFN-alpha/beta receptors than in wild-type mice, suggesting that IFN-alpha/beta is important in controlling replication. In contrast, IFN-gamma receptor-deficient mice infected with NA and SA strains had similar viremia levels and mortality rates to those of wild-type mice, suggesting that IFN-gamma does not play a major role in murine protection. Mice pretreated with poly(I-C), a nonspecific IFN inducer, exhibited dose-dependent protection against fatal eastern equine encephalitis, further evidence that IFN is important in controlling disease. Overall, our in vivo results did not support the hypothesis that NA strains are more virulent in humans due to their greater ability to counteract the IFN response. However, further studies using a better model of human disease are needed to confirm the results of differential human IFN sensitivity obtained in our in vitro experiments.

Replication and Clearance of Venezuelan Equine Encephalitis Virus from the Brains of Animals Vaccinated with Chimeric SIN/VEE Viruses

Venezuelan equine encephalitis virus (VEEV) is an important, naturally emerging zoonotic pathogen. Recent outbreaks in Venezuela and Colombia in 1995, involving an estimated 100,000 human cases, indicate that VEEV still poses a serious public health threat. To develop a safe, efficient vaccine that protects against disease resulting from VEEV infection, we generated chimeric Sindbis (SIN) viruses expressing structural proteins of different strains of VEEV and analyzed their replication in vitro and in vivo, as well as the characteristics of the induced immune responses. None of the chimeric SIN/VEE viruses caused any detectable disease in adult mice after either intracerebral (i.c.) or subcutaneous (s.c.) inoculation, and all chimeras were more attenuated than the vaccine strain, VEEV TC83, in 6-day-old mice after i.c. infection. All vaccinated mice were protected against lethal encephalitis following i.c., s.c., or intranasal (i.n.) challenge with the virulent VEEV ZPC738 strain (ZPC738). In spite of the absence of clinical encephalitis in vaccinated mice challenged with ZPC738 via i.n. or i.c. route, we regularly detected high levels of infectious challenge virus in the central nervous system (CNS). However, infectious virus was undetectable in the brains of all immunized animals at 28 days after challenge. Hamsters vaccinated with chimeric SIN/VEE viruses were also protected against s.c. challenge with ZPC738. Taken together, our findings suggest that these chimeric SIN/VEE viruses are safe and efficacious in adult mice and hamsters and are potentially useful as VEEV vaccines. In addition, immunized animals provide a useful model for studying the mechanisms of the anti-VEEV neuroinflammatory response, leading to the reduction of viral titers in the CNS and survival of animals.

Venezuelan Encephalitis Emergence Mediated by a Phylogenetically Predicted Viral Mutation

RNA viruses are notorious for their genetic plasticity and propensity to exploit new host-range opportunities, which can lead to the emergence of human disease epidemics such as severe acute respiratory syndrome, AIDS, dengue, and influenza. However, the mechanisms of host-range change involved in most of these viral emergences, particularly the genetic mechanisms of adaptation to new hosts, remain poorly understood. We studied the emergence of Venezuelan equine encephalitis virus (VEEV), an alphavirus pathogen of people and equines that has had severe health and economic effects in the Americas since the early 20th century. Between epidemics, VEE disappears for periods up to decades, and the viral source of outbreaks has remained enigmatic. Combined with phylogenetic analyses to predict mutations associated with a 1992-1993 epidemic, we used reverse genetic studies to identify an envelope glycoprotein gene mutation that mediated emergence. This mutation allowed an enzootic, equine-avirulent VEEV strain, which circulates among rodents in nearby forests to adapt for equine amplification. RNA viruses including alphaviruses exhibit high mutation frequencies. Therefore, ecological and epidemiological factors probably constrain the frequency of VEE epidemics more than the generation, via mutation, of amplification-competent (high equine viremia) virus strains. These results underscore the ability of RNA viruses to alter their host range, virulence, and epidemic potential via minor genetic changes. VEE also demonstrates the unpredictable risks to human health of anthropogenic changes such as the introduction of equines and humans into habitats that harbor zoonotic RNA viruses.

Reverse Transcription-PCR-enzyme-linked Immunosorbent Assay for Rapid Detection and Differentiation of Alphavirus Infections

Due to the lack of a rapid, simple, and inexpensive assay for detecting alphavirus infections, we combined a reverse transcription-PCR with an enzyme-linked immunosorbent assay (RT-PCR-ELISA) to identify human pathogenic alphaviruses that are endemic in the New World. By combining the sensitivity of PCR, the detection simplicity of ELISA, and the specificities of DNA probes, this method rapidly detected and differentiated closely related species and subtypes of several medically important alphaviruses. After an amplification using RT-PCR with primers targeting conserved sequences in the nonstructural protein 1 gene, sequence-specific, biotin-labeled probes targeted against Venezuelan, eastern, and western equine encephalitis or Mayaro virus genes were used for the detection of amplicons using ELISA. The assay is simple, fast, and easy to perform in an ordinary diagnostic laboratory or clinical setting. Nucleic acid derived from cell cultures infected with several alphaviruses, clinical specimens, and mosquito pools as well as frozen and paraffin-embedded animal tissues were detected and identified within 6 to 7 h in a sensitive and specific manner.

The Old World and New World Alphaviruses Use Different Virus-specific Proteins for Induction of Transcriptional Shutoff

Alphaviruses are widely distributed throughout the world. During the last few thousand years, the New World viruses, including Venezuelan equine encephalitis virus (VEEV) and eastern equine encephalitis virus (EEEV), evolved separately from those of the Old World, i.e., Sindbis virus (SINV) and Semliki Forest virus (SFV). Nevertheless, the results of our study indicate that both groups have developed the same characteristic: their replication efficiently interferes with cellular transcription and the cell response to virus replication. Transcriptional shutoff caused by at least two of the Old World alphaviruses, SINV and SFV, which belong to different serological complexes, depends on nsP2, but not on the capsid protein, functioning. Our data suggest that the New World alphaviruses VEEV and EEEV developed an alternative mechanism of transcription inhibition that is mainly determined by their capsid protein, but not by the nsP2. The ability of the VEEV capsid to inhibit cellular transcription appears to be controlled by the amino-terminal fragment of the protein, but not by its protease activity or by the positively charged RNA-binding domain. These data provide new insights into alphavirus evolution and present a plausible explanation for the particular recombination events that led to the formation of western equine encephalitis virus (WEEV) from SINV- and EEEV-like ancestors. The recombination allowed WEEV to acquire capsid protein functioning in transcription inhibition from EEEV-like virus. Identification of the new functions in the New World alphavirus-derived capsids opens an opportunity for developing new, safer alphavirus-based gene expression systems and designing new types of attenuated vaccine strains of VEEV and EEEV.

Recombinant Alphaviruses Are Safe and Useful Serological Diagnostic Tools

Serological assays for diagnosis of Venezuelan equine encephalitis virus (VEEV) currently require bio-safety level 3 facilities and select agent certification to produce antigens, reference sera, or viral stocks. Rapid identification of VEEV infection is required to respond to human and equine outbreaks of encephalitis caused by that virus and can be useful for epidemiologic surveillance. Alphavirus (Sindbis)-based recombinant viruses that express VEEV structural proteins are attenuated in animal models, thus representing an alternative to the handling of virulent infectious virus. Virus and viral antigens from recombinant Sindbis/VEE constructs engineered to express structural proteins from multiple VEEV subtypes were evaluated as diagnostic reagents in VEEV-specific serological assays, e.g., plaque reduction neutralization test (PRNT), hemagglutination inhibition (HI) assay, and complement fixation (CF) test. Chimeric viruses were produced efficiently in cell culture and were as effective as the parental virus for identifying infection of humans, horses, and rodents in these serological assays.

Comparative Analysis of the Alphavirus-based Vectors Expressing Rift Valley Fever Virus Glycoproteins

During the last decade, alphaviruses became widely used for expression of heterologous genetic information and development of recombinant vaccines against a variety of human and animal pathogens. In this study, we compared a number of vectors based on the genome of Sindbis (SINV) and Venezuelan equine encephalitis (VEEV) viruses for their ability to express the Rift Valley fever virus (RVFV) envelope glycoprotein Gn and induce a protective immune response against RVFV infection. Our results suggest that (i) application of VEEV-based expression systems appears to be advantageous, when compared to similar systems designed on the basis of the SINV genome. (ii) Alphavirus-specific E3 and E2 proteins and furin-specific cleavage sites can be used for engineering secreted forms of the proteins. (iii) Alphaviruses can be modified for expression of the large fragments of heterologous proteins on the surface of chimeric, infectious viral particles. Thus, alphavirus-based expression systems may have the potential for a broader application beyond their current use as replicons or double-subgenomic vectors.

Alpha-beta T Cells Provide Protection Against Lethal Encephalitis in the Murine Model of VEEV Infection

We evaluated the safety and immunogenicity of a chimeric alphavirus vaccine candidate in mice with selective immunodeficiencies. This vaccine candidate was highly attenuated in mice with deficiencies in the B and T cell compartments, as well as in mice with deficient gamma-interferon responsiveness. However, the level of protection varied among the strains tested. Wild type mice were protected against lethal VEEV challenge. In contrast, alpha/beta (alphabeta) TCR-deficient mice developed lethal encephalitis following VEEV challenge, while mice deficient in gamma/delta (gammadelta) T cells were protected. Surprisingly, the vaccine potency was diminished by 50% in animals lacking interferon-gamma receptor alpha chain (R1)-chain and a minority of vaccinated immunoglobulin heavy chain-deficient (microMT) mice survived challenge, which suggests that neutralizing antibody may not be absolutely required for protection. Prolonged replication of encephalitic VEEV in the brain of pre-immunized mice is not lethal and adoptive transfer experiments indicate that CD3(+) T cells are required for protection.

Chimeric Sindbis/eastern Equine Encephalitis Vaccine Candidates Are Highly Attenuated and Immunogenic in Mice

We developed chimeric Sindbis (SINV)/eastern equine encephalitis (EEEV) viruses and investigated their potential for use as live virus vaccines against EEEV. One vaccine candidate contained structural protein genes from a typical North American EEEV strain, while the other had structural proteins from a naturally attenuated Brazilian isolate. Both chimeric viruses replicated efficiently in mammalian and mosquito cell cultures and were highly attenuated in mice. Vaccinated mice did not develop detectable disease or viremia, but developed high titers of neutralizing antibodies. Upon challenge with EEEV, mice vaccinated with >10(4) PFU of the chimeric viruses were completely protected from disease. These findings support the potential use of these SIN/EEEV chimeras as safe and effective vaccines.

Protection of Mice by Equine Herpesvirus Type 1 Based Experimental Vaccine Against Lethal Venezuelan Equine Encephalitis Virus Infection in the Absence of Neutralizing Antibodies

A vectored vaccine based on equine herpesvirus type 1 (EHV-1) was generated as an alternative for safe and efficient prophylaxis against Venezuelan equine encephalitis virus (VEEV) infection. Two-step (en passant) Red mutagenesis was used to insert VEEV structural genes into an infectious clone of EHV-1 vaccine strain RacH. The recombinant virus, rH_VEEV, efficiently and stably expressed VEEV structural proteins as detected by various antibodies, including a conformation-dependent monoclonal antibody to envelope glycoprotein E2. In addition, rH_VEEV was indistinguishable from parental bacterial artificial chromosome-derived virus with respect to growth properties in cultured cells. Immunization of mice with the vectored vaccine conferred full protection against lethal challenge infection using VEEV strain ZPC738 in the absence of neutralizing antibodies and in a dose-dependent manner. Analyses of IgG responses demonstrated production of VEEV-specific IgG1 and total IgG antibodies after vaccination, indicating that protection was dependent on either cytotoxic T cell responses or antibody-mediated protection unrelated to neutralizing activity.

Injectable Peramivir Mitigates Disease and Promotes Survival in Ferrets and Mice Infected with the Highly Virulent Influenza Virus, A/Vietnam/1203/04 (H5N1)

The post-exposure therapeutic efficacy of injectable peramivir against highly pathogenic avian influenza type A H5N1 was evaluated in mice and in ferrets. Seventy to eighty percent of the H5N1-infected peramivir-treated mice, and 70% in the oseltamivir treated mice survived the 15-day study period, as compared to 36% in control (vehicle) group. Ferrets were infected intranasally with H5N1 followed by treatment with multiple doses of peramivir. In two of three trials, a statistically significant increase in survival over a 16-18 day period resulted from peramivir treatment, with improved survival of 40-64% in comparison to mock-treated or untreated animals. Injected peramivir mitigates virus-induced disease, reduces infectious virus titers in the lungs and brains and promotes survival in ferrets infected intranasally with this highly neurovirulent isolate. A single intramuscular peramivir injection protected mice against severe disease outcomes following infection with highly pathogenic avian influenza and multi-dose treatment was efficacious in ferrets.

Novel Linear DNA Vaccines Induce Protective Immune Responses Against Lethal Infection with Influenza Virus Type A/H5N1

Vaccine development for possible influenza pandemics has been challenging. Conventional vaccines such as inactivated and live attenuated virus preparations are limited in terms of production speed and capacity. DNA vaccination has emerged as a potential alternative to conventional vaccines against influenza pandemics. In this study, we use a novel, cell-free DNA manufacturing process (synDNA) to produce prototype linear DNA vaccines against the influenza virus type A/H5N1. This synDNA process does not require bacterial fermentation, so it avoids the use of antibiotic resistance genes and other nucleic acid sequences unrelated to the antigen gene expression in the actual therapeutic DNA construct. The efficacy of various vaccines expressing the hemagglutinin and neuraminidase proteins (H5N1 synDNA), hemagglutinin alone (H5 synDNA) or neuraminidase alone (N1 synDNA) was evaluated in mice. Two of the constructs (H5 synDNA and H5N1 synDNA) induced a robust protective immune response with up to 93% of treated mice surviving a lethal challenge of a virulent influenza A/Vietnam/1203/04 H5N1 isolate. In combination with a potent biological activity and simplified production footprint, these characteristics make DNA vaccines prepared with our synDNA process highly suitable as alternatives to other vaccine preparations.

Inhibition of Alphavirus Infection in Cell Culture and in Mice with Antisense Morpholino Oligomers

The genus Alphavirus contains members that threaten human health, both as natural pathogens and as potential biological weapons. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) enter cells readily and can inhibit viral replication through sequence-specific steric blockade of viral RNA. Sindbis virus (SINV) has low pathogenicity in humans and is regularly utilized as a model alphavirus. PPMO targeting the 5'-terminal and AUG translation start site regions of the SINV genome blocked the production of infectious SINV in tissue culture. PPMO designed against corresponding regions in Venezuelan equine encephalitis virus (VEEV) were likewise found to be effective in vitro against several strains of VEEV. Mice treated with PPMO before and after VEEV infection were completely protected from lethal outcome while mice receiving only post-infection PPMO treatment were partially protected. Levels of virus in tissue samples correlated with animal survival. Uninfected mice suffered no apparent ill-effects from PPMO treatment. Thus, PPMO appear promising as candidates for therapeutic development against alphaviruses.

Pathogenesis of XJ and Romero Strains of Junin Virus in Two Strains of Guinea Pigs

Argentine hemorrhagic fever (AHF), a systemic infectious disease caused by infection with Junin virus, affects several organs, and patients can show hematologic, cardiovascular, renal, or neurologic symptoms. We compared the virulence of two Junin virus strains in inbred and outbred guinea pigs with the aim of characterizing this animal model better for future vaccine/antiviral efficacy studies. Our data indicate that this passage of the XJ strain is attenuated in guinea pigs. In contrast, the Romero strain is highly virulent in Strain 13 as well as in Hartley guinea pigs, resulting in systemic infection, thrombocytopenia, elevated aspartate aminotransferase levels, and ultimately, uniformly lethal disease. We detected viral antigen in formalin-fixed, paraffin-embedded tissues. Thus, both guinea pig strains are useful animal models for lethal Junin virus (Romero strain) infection and potentially can be used for preclinical trials in vaccine or antiviral drug development.

Host Immunity in the Protective Response to Vaccination with Heat-killed Burkholderia Mallei

We performed initial cell, cytokine and complement depletion studies to investigate the possible role of these effectors in response to vaccination with heat-killed Burkholderia mallei in a susceptible BALB/c mouse model of infection.

Susceptibility of the Aotus Nancymaae Owl Monkey to Eastern Equine Encephalitis

Eastern equine encephalitis virus (EEEV) is an arthropod-borne virus associated with life-threatening encephalitis in humans, equines, birds and many other domestic animals. To investigate the suitability of the Aotus nancymaae New World owl monkey as a viable animal model for EEE candidate vaccine testing we used clinical presentation, serology, viral isolation and PCR to evaluate pathogenesis and immunity in infected animals. Monkeys were inoculated subcutaneously (SQ) or intranasally (IN) with 10(4)pfu of virulent EEEV and were initially followed for 45 days. While none of the animals displayed clinical signs of disease, all of the SC inoculated animals (n=6) manifested a viremia averaging 3.2 days (+/-0.8 days). Likewise, serologic responses (IgM, IgG and PRNT) were observed in all SC infected animals. Interestingly, none of the IN inoculated animals (n=6) became viremic or mounted an antibody response and no pathological abnormalities were observed in two animals that were necropsied on day 6 post-infection (p.i.) from each group. To determine if the antibodies produced by the SC inoculated animals were protective against homologous challenge, three animals from the SC group were serologically evaluated on day 253 p.i. and were administered an inoculum identical to initial challenge on day 270 p.i. A positive control group of four naïve animals was also infected as before. All of the naïve positive control animals manifested a similar viremia as observed initially, averaging 2.75 days (+/-0.5 days) while none of the previously challenged animals became viremic. On days 45 and 253 p.i. geometric mean PRNT titers in the SC group were 453 and 101, respectively. This study demonstrates that the Aotus nancymaae can be reproducibly infected with EEE virus and can serve as a suitable model for infection and immunogenicity for the evaluation of candidate vaccines against EEEV.

CD4+ T Cells Provide Protection Against Acute Lethal Encephalitis Caused by Venezuelan Equine Encephalitis Virus

Studying the mechanisms of host survival resulting from viral encephalitis is critical to the development of vaccines. Here we have shown in several independent studies that high dose treatment with neutralizing antibody prior to intranasal infection with Venezuelan equine encephalitis virus had an antiviral effect in the visceral organs and prolonged survival time of infected mice, even in the absence of alphabeta T cells. Nevertheless, antibody treatment did not prevent the development of lethal encephalitis. On the contrary, the adoptive transfer of primed CD4(+) T cells was necessary to prevent lethal encephalitis in mice lacking alphabeta T cell receptor.

Superior Efficacy of a Recombinant Flagellin:H5N1 HA Globular Head Vaccine is Determined by the Placement of the Globular Head Within Flagellin

Transmission of highly pathogenic avian influenza (HPAI) between birds and humans is an ongoing threat that holds potential for the emergence of a pandemic influenza strain. A major barrier to an effective vaccine against avian influenza has been the generally poor immunopotency of many of the HPAI strains coupled with the manufacturing constraints employing conventional methodologies. Fusion of flagellin, a toll-like receptor-5 ligand, to vaccine antigens has been shown to enhance the immune response to the fused antigen in preclinical studies. Here, we have evaluated the immunogenicity and efficacy of a panel of flagellin-based hemagglutinin (HA) globular head fusion vaccines in inbred mice. The HA globular head of these vaccines is derived from the A/Vietnam/1203/04 (VN04; H5N1) HA molecule. We find that replacement of domain D3 of flagellin with the VN04 HA globular head creates a highly effective vaccine that elicits protective HAI titers which protect mice against disease and death in a lethal challenge model.

Vaccines for Venezuelan Equine Encephalitis

Arboviruses are capable of causing encephalitis in animals and human population when transmitted by the vector or potentially via infectious aerosol. Recent re-emergence of Venezuelan equine encephalitis virus (VEEV) in South America emphasizes the importance of this pathogen to public health and veterinary medicine. Despite its importance no antivirals or vaccines against VEEV are currently available in the USA. Here we review some of the older and newer approaches aimed at generating a safe and immunogenic vaccine as well as most recent data about the mechanistic of protection in animal models of infection.

Encephalitic Alphaviruses

This review will cover zoonotic, encephalitic alphaviruses in the family Togaviridae. Encephalitic alphaviruses, i.e. Western- (WEEV), Eastern- (EEEV), Venezuelan equine encephalitis virus (VEEV) and, more rarely, Ross River virus, Chikungunya virus and Highlands J virus (HJV), are neuroinvasive and may cause neurological symptoms ranging from mild (e.g., febrile illness) to severe (e.g., encephalitis) in humans and equines. Among the naturally occurring alphaviruses, WEEV, EEEV and VEEV have widespread distributions in North, Central and South America. WEEV has found spanning the U.S. from the mid-West (Michigan and Illinois) to the West coast and extending to Canada with human cases reported in 21 states. EEEV is found along the Gulf (Texas to Florida) and Atlantic Coast (Georgia to New Hampshire), as well as in the mid-West (Wisconsin, Illinois and Michigan) and in Canada, with human cases reported in 19 states. In contrast, transmission of VEEV occurs predominantly in Central and South America. As with their geographical distribution, equine encephalitis viruses differ in their main mosquito vector species and their zoonotic potential.

TC83 Replicon Vectored Vaccine Provides Protection Against Junin Virus in Guinea Pigs

Junin virus (JUNV) is the etiological agent of the potentially lethal, reemerging human disease, Argentine hemorrhagic fever (AHF). The mechanism of the disease development is not well understood and no antiviral therapy is available. Candid 1, a live-attenuated vaccine, has been developed by the US Army and is being used in the endemic area to prevent AHF. This vaccine is only approved for use in Argentina. In this study we have used the alphavirus-based approach to engineer a replicon system based on a human (United States Food and Drug Administration Investigational New Drug status) vaccine TC83 that express heterologous viral antigens, such as glycoproteins (GPC) of Junin virus (JUNV). Preclinical studies testing the immunogenicity and efficacy of TC83/GPC were performed in guinea pigs. A single dose of the live-attenuated alphavirus based vaccine expressing only GPC was immunogenic and provided partial protection, while a double dose of the same vaccine provided a complete protection against JUNV. This is the first scientific report to our knowledge that the immune response against GPC alone is sufficient to prevent lethal disease against JUNV in an animal model.

Mice Lacking Alpha/beta and Gamma Interferon Receptors Are Susceptible to Junin Virus Infection

Junin virus (JUNV) causes a highly lethal human disease, Argentine hemorrhagic fever. Previous work has demonstrated the requirement for human transferrin receptor 1 for virus entry, and the absence of the receptor was proposed to be a major cause for the resistance of laboratory mice to JUNV infection. In this study, we present for the first time in vivo evidence that the disruption of interferon signaling is sufficient to generate a disease-susceptible mouse model for JUNV infection. After peripheral inoculation with virulent JUNV, adult mice lacking alpha/beta and gamma interferon receptors developed disseminated infection and severe disease.

Antiviral Activity of a Small-molecule Inhibitor of Arenavirus Glycoprotein Processing by the Cellular Site 1 Protease

Arenaviruses merit interest as clinically important human pathogens and include several causative agents, chiefly Lassa virus (LASV), of hemorrhagic fever disease in humans. There are no licensed LASV vaccines, and current antiarenavirus therapy is limited to the use of ribavirin, which is only partially effective and is associated with significant side effects. The arenavirus glycoprotein (GP) precursor GPC is processed by the cellular site 1 protease (S1P) to generate the peripheral virion attachment protein GP1 and the fusion-active transmembrane protein GP2, which is critical for production of infectious progeny and virus propagation. Therefore, S1P-mediated processing of arenavirus GPC is a promising target for therapeutic intervention. To this end, we have evaluated the antiarenaviral activity of PF-429242, a recently described small-molecule inhibitor of S1P. PF-429242 efficiently prevented the processing of GPC from the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) and LASV, which correlated with the compound's potent antiviral activity against LCMV and LASV in cultured cells. In contrast, a recombinant LCMV expressing a GPC whose processing into GP1 and GP2 was mediated by furin, instead of S1P, was highly resistant to PF-429242 treatment. PF-429242 did not affect virus RNA replication or budding but had a modest effect on virus cell entry, indicating that the antiarenaviral activity of PF-429242 was mostly related to its ability to inhibit S1P-mediated processing of arenavirus GPC. Our findings support the feasibility of using small-molecule inhibitors of S1P-mediated processing of arenavirus GPC as a novel antiviral strategy.

Rescue from Cloned CDNAs and in Vivo Characterization of Recombinant Pathogenic Romero and Live-attenuated Candid #1 Strains of Junin Virus, the Causative Agent of Argentine Hemorrhagic Fever Disease

The New World arenavirus Junin virus (JUNV) is the causative agent of Argentine hemorrhagic fever (AHF), which is associated with high morbidity and significant mortality. Several pathogenic strains of JUNV have been documented, and a highly attenuated vaccine strain (Candid #1) was generated and used to vaccinate the human population at risk. The identification and functional characterization of viral genetic determinants associated with AHF and Candid #1 attenuation would contribute to the elucidation of the mechanisms contributing to AHF and the development of better vaccines and therapeutics. To this end, we used reverse genetics to rescue the pathogenic Romero and the attenuated Candid #1 strains of JUNV from cloned cDNAs. Both recombinant Candid #1 (rCandid #1) and Romero (rRomero) had the same growth properties and phenotypic features in cultured cells and in vivo as their corresponding parental viruses. Infection with rRomero caused 100% lethality in guinea pigs, whereas rCandid #1 infection was asymptomatic and provided protection against a lethal challenge with Romero. Notably, Romero and Candid #1 trans-acting proteins, L and NP, required for virus RNA replication and gene expression were exchangeable in a minigenome rescue assay. These findings support the feasibility of studies aimed at determining the contribution of each viral gene to JUNV pathogenesis and attenuation. In addition, we rescued Candid #1 viruses with three segments that efficiently expressed foreign genes introduced into their genomes. This finding opens the way for the development of a safe multivalent arenavirus vaccine.

Prevention of Influenza Virus Shedding and Protection from Lethal H1N1 Challenge Using a Consensus 2009 H1N1 HA and NA Adenovirus Vector Vaccine

Vaccines against emerging pathogens such as the 2009 H1N1 pandemic virus can benefit from current technologies such as rapid genomic sequencing to construct the most biologically relevant vaccine. A novel platform (Ad5 [E1-, E2b-]) has been utilized to induce immune responses to various antigenic targets. We employed this vector platform to express hemagglutinin (HA) and neuraminidase (NA) genes from 2009 H1N1 pandemic viruses. Inserts were consensuses sequences designed from viral isolate sequences and the vaccine was rapidly constructed and produced. Vaccination induced H1N1 immune responses in mice, which afforded protection from lethal virus challenge. In ferrets, vaccination protected from disease development and significantly reduced viral titers in nasal washes. H1N1 cell mediated immunity as well as antibody induction correlated with the prevention of disease symptoms and reduction of virus replication. The Ad5 [E1-, E2b-] should be evaluated for the rapid development of effective vaccines against infectious diseases.

Rapid, Non-invasive Imaging of Alphaviral Brain Infection: Reducing Animal Numbers and Morbidity to Identify Efficacy of Potential Vaccines and Antivirals

Rapid and accurate identification of disease progression are key factors in testing novel vaccines and antivirals against encephalitic alphaviruses. Typical efficacy studies utilize a large number of animals and severe morbidity or mortality as an endpoint. New technologies provide a means to reduce and refine the animal use as proposed in Hume's 3Rs (replacement, reduction, refinement) described by Russel and Burch. In vivo imaging systems (IVIS) and bioluminescent enzyme technologies accomplish the reduction of animal requirements while shortening the experimental time and improving the accuracy in localizing active virus replication. In the case of murine models of viral encephalitis in which central nervous system (CNS) viral invasion occurs rapidly but the disease development is relatively slow, we visualized the initial brain infection and enhance the data collection process required for efficacy studies on antivirals or vaccines that are aimed at preventing brain infection. Accordingly, we infected mice through intranasal inoculation with the genetically modified pathogen, Venezuelan equine encephalitis, which expresses a luciferase gene. In this study, we were able to identify the invasion of the CNS at least 3 days before any clinical signs of disease, allowing for reduction of animal morbidity providing a humane means of disease and vaccine research while obtaining scientific data accurately and more rapidly. Based on our data from the imaging model, we confirmed the usefulness of this technology in preclinical research by demonstrating the efficacy of Ampligen, a TLR-3 agonist, in preventing CNS invasion.

Vγ4+ T Cells Regulate Host Immune Response to West Nile Virus Infection

The Vγ4(+) cells, a subpopulation of peripheral γδ T cells, are involved in West Nile virus (WNV) pathogenesis, but the underlying mechanism remains unclear. In this study, we found that WNV-infected Vγ4(+) cell-depleted mice had lower viremia and a reduced inflammatory response in the brain. The Vγ4(+) cells produced IL-17 during WNV infection, but blocking IL-17 signaling did not affect host susceptibility to WNV encephalitis. We also noted that there was an enhanced magnitude of protective splenic Vγ1(+) cell expansion in Vγ4(+) cell-depleted mice compared to that in controls during WNV infection. In addition, Vγ4(+) cells of WNV-infected mice had a higher potential for producing TGF-β. The γδ T cells of WNV-infected Vγ4(+) cell-depleted mice had a higher proliferation rate than those of WNV-infected controls upon ex vivo stimulation with anti-CD3, and this difference was diminished in the presence of TGF-β inhibitor. Finally, Vγ4(+) cells of infected mice contributed directly and indirectly to the higher level of IL-10, which is known to play a negative role in immunity against WNV infection. In summary, Vγ4(+) cells suppress Vγ1(+) cell expansion via TGF-β and increase IL-10 level during WNV infection, which together may lead to higher viremia and enhanced brain inflammation.

Neuropathology of H5N1 Virus Infection in Ferrets

Highly pathogenic H5N1 virus remains a potential threat to humans. Over 289 fatalities have been reported in WHO confirmed human cases since 2003, and lack of effective vaccines and early treatments contribute to increasing numbers of cases and fatalities. H5N1 encephalitis is a recognized cause of death in Vietnamese cases, and brain pathology is described in other human cases and naturally infected animals. However, neither pathogenesis of H5N1 viral infection in human brain nor post-infection effects in survivors have been fully investigated. We report the brain pathology in a ferret model for active infection and 18-day survival stages. This model closely resembles the infection pattern and progression in human cases of influenza A, and our report is the first description of brain pathology for longer term (18-day) survival in ferrets. We analyzed viral replication, type and severity of meningoencephalitis, infected cell types, and cellular responses to infection. We found viral replication to very high titers in ferret brain, closely correlating with severity of meningoencephalitis. Viral antigens were detected predominantly in neurons, correlating with inflammatory lesions, and less frequently in astrocytes and ependymal cells during active infection. Mononuclear cell infiltrates were observed in early stages predominantly in cerebral cortex, brainstem, and leptomeninges, and less commonly in cerebellum and other areas. Astrogliosis was mild at day 4 post-infection, but robust by day 18. Early and continuous treatment with an antiviral agent (peramivir) inhibited virus production to non-detectable levels, reduced severity of brain injury, and promoted higher survival rates.

Functional Interferon System is Required for Clearance of Lassa Virus

Lassa virus (LASV) is the causative agent of Lassa hemorrhagic fever (LF) in humans, a deadly disease endemic to West Africa that results in 5,000 to 10,000 deaths annually. Here we present results demonstrating that functional type I and type II interferon (IFN) signaling is required for efficient control of LASV dissemination and clearance.

Natural Killer Cell Mediated Pathogenesis Determines Outcome of Central Nervous System Infection with Venezuelan Equine Encephalitis Virus in C3H/HeN Mice

TC83 is a human vaccine with investigational new drug status and is used as a prototype Venezuelan equine encephalitis virus for pathogenesis and antiviral research. Differing from other experimental models, the virus causes high titer infection in the brain and 90-100% mortality in the C3H/HeN murine model. To better characterize the susceptibility to disease development in C3H/HeN mice, we have analyzed the gene transcriptomes and cytokine production in the brains of infected mice. Our analysis indicated the potential importance of natural killer cells in the encephalitic disease development. This paper describes for the first time a pathogenic role for natural killer cells in VEEV encephalitis.

Junín Virus Infection Activates the Type I Interferon Pathway in a RIG-I-dependent Manner

Junín virus (JUNV), an arenavirus, is the causative agent of Argentine hemorrhagic fever, an infectious human disease with 15-30% case fatality. The pathogenesis of AHF is still not well understood. Elevated levels of interferon and cytokines are reported in AHF patients, which might be correlated to the severity of the disease. However the innate immune response to JUNV infection has not been well evaluated. Previous studies have suggested that the virulent strain of JUNV does not induce IFN in human macrophages and monocytes, whereas the attenuated strain of JUNV was found to induce IFN response in murine macrophages via the TLR-2 signaling pathway. In this study, we investigated the interaction between JUNV and IFN pathway in human epithelial cells highly permissive to JUNV infection. We have determined the expression pattern of interferon-stimulated genes (ISGs) and IFN-β at both mRNA and protein levels during JUNV infection. Our results clearly indicate that JUNV infection activates the type I IFN response. STAT1 phosphorylation, a downstream marker of activation of IFN signaling pathway, was readily detected in JUNV infected IFN-competent cells. Our studies also demonstrated for the first time that RIG-I was required for IFN production during JUNV infection. IFN activation was detected during infection by either the virulent or attenuated vaccine strain of JUNV. Curiously, both virus strains were relatively insensitive to human IFN treatment. Our studies collectively indicated that JUNV infection could induce host type I IFN response and provided new insights into the interaction between JUNV and host innate immune system, which might be important in future studies on vaccine development and antiviral treatment.

Resequencing of the Puumala Virus Strain Sotkamo from the WHO Arbovirus Collection

RNA viruses exhibit a high mutation rate as the RNA-dependent RNA polymerase lacks proofreading and repair capabilities. It is known that serial passaging on cell culture leads to virus adaptation. Puumala virus (PUUV) strain Sotkamo is the prototype virus for the low-pathogenic hantavirus Puumala, family Bunyaviridae. A full-length sequence of the strain Sotkamos tripartite genome was made available more than 15 years ago, after at least 15 passages on Vero E6 cells. A distinct sample from the sequenced strain, with unknown passage history, was then included in the WHO Arbovirus collection. The genome sequence of this included sample was determined in this study and exhibited over 99 % identity in comparison to the previously published sequence. A total of 23 nucleotide changes across all genome segments were found. The small segment had the highest nucleotide variance without changes on the protein level. Within the extraviral domain of the glycoproteins, the majority of non-synonymous mutations were detected, whereas the large segment is most conserved on the nucleotide level. It seems possible that the PUUV strain Sotkamo adapted differently to serial passaging on cell culture in two different laboratories. In addition, a distinct passage number could exhibit itself within the nucleotide differences.

Control of SIV Infection and Subsequent Induction of Pandemic H1N1 Immunity in Rhesus Macaques Using an Ad5 [E1-, E2b-] Vector Platform

Anti-vector immunity mitigates immune responses induced by recombinant adenovirus vector vaccines, limiting their prime-boost capabilities. We have developed a novel gene delivery and expression platform (Ad5 [E1-, E2b-]) that induces immune responses despite pre-existing and/or developed concomitant Ad5 immunity. In the present study, we evaluated if this new Ad5 platform could overcome the adverse condition of pre-existing Ad5 immunity to induce effective immune responses in prime-boost immunization regimens against two different infectious diseases in the same animal. Ad5 immune rhesus macaques (RM) were immunized multiple times with the Ad5 [E1-, E2b-] platform expressing antigens from simian immunodeficiency virus (SIV). Immunized RM developed cell-mediated immunity against SIV antigens Gag, Pol, Nef and Env as well as antibody against Env. Vaccinated and vector control RMs were challenged intra-rectally with homologous SIVmac239. During a 7-week follow-up, there was perturbation of SIV load in some immunized RM. At 7 weeks post-challenge, eight immunized animals (53%) did not have detectable SIV, compared to two RM controls (13%) (P<0.02; log-rank Mantel-Cox test). There was no correlation of protective MHC contributing to infection control. The RM without detectable circulating SIV, now hyper immune to Ad5, were then vaccinated with the same Ad5 [E1-, E2b-] platform expressing H1N1 influenza hemagglutinin (HA). Thirty days post Ad5 [E1-, E2b-]-HA vaccination, significant levels of influenza neutralizing antibody were induced in all animals that increased after an Ad5 [E1-, E2b-]-HA homologous boost. These data demonstrate the versatility of this new vector platform to immunize against two separate disease targets in the same animal despite the presence of immunity against the delivery platform, permitting homologous repeat immunizations with an Ad5 gene delivery platform.

Pathogenesis of the Viral Hemorrhagic Fevers

Four families of enveloped RNA viruses, filoviruses, flaviviruses, arenaviruses, and bunyaviruses, cause hemorrhagic fevers. These viruses are maintained in specific natural cycles involving nonhuman primates, bats, rodents, domestic ruminants, humans, mosquitoes, and ticks. Vascular instability varies from mild to fatal shock, and hemorrhage ranges from none to life threatening. The pathogenic mechanisms are extremely diverse and include deficiency of hepatic synthesis of coagulation factors owing to hepatocellular necrosis, cytokine storm, increased permeability by vascular endothelial growth factor, complement activation, and disseminated intravascular coagulation in one or more hemorrhagic fevers. The severity of disease caused by these agents varies tremendously; there are extremely high fatality rates in Ebola and Marburg hemorrhagic fevers, and asymptomatic infection predominates in yellow fever and dengue viral infections. Although ineffective immunity and high viral loads are characteristic of several viral hemorrhagic fevers, severe plasma leakage occurs at the time of viral clearance and defervescence in dengue hemorrhagic fever. Expected final online publication date for the Annual Review of Pathology: Mechanisms of Disease Volume 8 is January 24, 2013. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.

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