Pregnancy and pandemics: Interaction of viral surface proteins and placenta cells.

Throughout history, pandemics of infectious diseases caused by emerging viruses have spread worldwide. Evidence from previous outbreaks demonstrated that pregnant women are at high risk of contracting the diseases and suffering from adverse outcomes. However, while some viruses can cause major health complications for the mother and her fetus, others do not appear to affect pregnancy. Viral surface proteins bind to specific receptors on the cellular membrane of host cells and begin therewith the infection process. During pregnancy, the molecular features of these proteins may determine specific target cells in the placenta, which may explain the different outcomes. In this review, we display information on Variola, Influenza, Zika and Corona viruses focused on their surface proteins, effects on pregnancy, and possible target placental cells. This will contribute to understanding viral entry during pregnancy, as well as to develop strategies to decrease the incidence of obstetrical problems in current and future infections.

Use of live Variola virus to determine whether CAST/EiJ mice are a suitable surrogate animal model for human smallpox.

Numerous animal models of systemic orthopoxvirus disease have been developed to evaluate therapeutics against variola virus (VARV), the causative agent of smallpox. These animal models do not resemble the disease presentation in human smallpox and most used surrogate Orthopoxviruses. A rodent model using VARV has a multitude of advantages, and previous investigations identified the CAST/EiJ mouse as highly susceptible to monkeypox virus infection, making it of interest to determine if these rodents are also susceptible to VARV infection. In this study, we inoculated CAST/EiJ mice with a range of VARV doses (102-106 plaque forming units). Some animals had detectable viable VARV from the oropharynx between days 3 and 12 post inoculation. Despite evidence of disease, the CAST/EiJ mouse does not provide a model for clinical smallpox due to mild signs of morbidity and limited skin lesions. However, in contrast to previous rodent models using VARV challenge (i.e. prairie dogs and SCID mice), a robust immune response was observed in the CAST/EiJ mice (measured by Immunoglobulin G enzyme-linked immunosorbent assay). This is an advantage of this model for the study of VARV and presents a unique potential for the study of the immunomodulatory pathways following VARV infection.

[We should be prepared to smallpox re-emergence.]

The review contains a brief analysis of the results of investigations conducted during 40 years after smallpox eradication and directed to study genomic organization and evolution of variola virus (VARV) and development of modern diagnostics, vaccines and chemotherapies of smallpox and other zoonotic orthopoxviral infections of humans. Taking into account that smallpox vaccination in several cases had adverse side effects, WHO recommended ceasing this vaccination after 1980 in all countries of the world. The result of this decision is that the mankind lost the collective immunity not only to smallpox, but also to other zoonotic orthopoxvirus infections. The ever more frequently recorded human cases of zoonotic orthopoxvirus infections force to renew consideration of the problem of possible smallpox reemergence resulting from natural evolution of these viruses. Analysis of the available archive data on smallpox epidemics, the history of ancient civilizations, and the newest data on the evolutionary relationship of orthopoxviruses has allowed us to hypothesize that VARV could have repeatedly reemerged via evolutionary changes in a zoonotic ancestor virus and then disappeared because of insufficient population size of isolated ancient civilizations. Only the historically last smallpox pandemic continued for a long time and was contained and stopped in the 20th century thanks to the joint efforts of medics and scientists from many countries under the aegis of WHO. Thus, there is no fundamental prohibition on potential reemergence of smallpox or a similar human disease in future in the course of natural evolution of the currently existing zoonotic orthopoxviruses. Correspondingly, it is of the utmost importance to develop and widely adopt state-of-the-art methods for efficient and rapid species-specific diagnosis of all orthopoxvirus species pathogenic for humans, VARV included. It is also most important to develop new safe methods for prevention and therapy of human orthopoxvirus infections.

Bayesian Phylogeography and Pathogenic Characterization of Smallpox Based on HA, ATI, and CrmB Genes.

Variola virus is at risk of re-emergence either through accidental release, bioterrorism, or synthetic biology. The use of phylogenetics and phylogeography to support epidemic field response is expected to grow as sequencing technology becomes miniaturized, cheap, and ubiquitous. In this study, we aimed to explore the use of common VARV diagnostic targets hemagglutinin (HA), cytokine response modifier B (CrmB), and A-type inclusion protein (ATI) for phylogenetic characterization as well as the representativeness of modelling strategies in phylogeography to support epidemic response should smallpox re-emerge. We used Bayesian discrete-trait phylogeography using the most complete data set currently available of whole genome (n = 51) and partially sequenced (n = 20) VARV isolates. We show that multilocus models combining HA, ATI, and CrmB genes may represent a useful heuristic to differentiate between VARV Major and subclades of VARV Minor which have been associated with variable case-fatality rates. Where whole genome sequencing is unavailable, phylogeography models of HA, ATI, and CrmB may provide preliminary but uncertain estimates of transmission, while supplementing whole genome models with additional isolates sequenced only for HA can improve sample representativeness, maintaining similar support for transmission relative to whole genome models. We have also provided empirical evidence delineating historic international VARV transmission using phylogeography. Due to the persistent threat of re-emergence, our results provide important research for smallpox epidemic preparedness in the posteradication era as recommended by the World Health Organisation.

Dynamics of Pathological and Virological Findings During Experimental Calpox Virus Infection of Common Marmosets (Callithrix jacchus).

Experimental intranasal infection of marmosets (Callithrix jacchus) with calpox virus results in fatal disease. Route and dose used for viral inoculation of the test animals mimics the natural transmission of smallpox, thus representing a suitable model to study pathogenesis and to evaluate new vaccines against orthopoxvirus infection. However, the pathogenic mechanisms leading to death are still unclear. Therefore, our study aimed at investigating the kinetics of pathological alterations to clarify the pathogenesis in calpox virus infection. Following intranasal inoculation with two different viral doses, common marmosets were sacrificed on days 3, 5, 7, 10 and 12 post inoculation. Collected tissue was screened using histopathology, immunohistochemistry, transmission electron microscopy, and virological assays. Our data suggest that primary replication took place in nasal and bronchial epithelia followed by secondary replication in submandibular lymph nodes and spleen. Parallel to viremia at day 7, virus was detectable in many organs, mainly located in epithelial cells and macrophages, as well as in endothelial cells. Based on the onset of clinical signs, the histological and ultrastructural lesions and the immunohistochemical distribution pattern of the virus, the incubation period was defined to last 11 days, which resembles human smallpox. In conclusion, the data indicate that the calpox model is highly suitable for studying orthopoxvirus-induced disease.

Re-Assembly and Analysis of an Ancient Variola Virus Genome.

We report a major improvement to the assembly of published short read sequencing data from an ancient variola virus (VARV) genome by the removal of contig-capping sequencing tags and manual searches for gap-spanning reads. The new assembly, together with camelpox and taterapox genomes, permitted new dates to be calculated for the last common ancestor of all VARV genomes. The analysis of recently sequenced VARV-like cowpox virus genomes showed that single nucleotide polymorphisms (SNPs) and amino acid changes in the vaccinia virus (VACV)-Cop-O1L ortholog, predicted to be associated with VARV host specificity and virulence, were introduced into the lineage before the divergence of these viruses. A comparison of the ancient and modern VARV genome sequences also revealed a measurable drift towards adenine + thymine (A + T) richness.

Variola minor in coalfield areas of England and Wales, 1921-34: Geographical determinants of a national smallpox epidemic that spread out of effective control.

This paper uses techniques of binary logistic regression to identify the spatial determinants of the last national epidemic of smallpox to spread in England and Wales, the variola minor epidemic of 1921-34. Adjusting for age and county-level variations in vaccination coverage in infancy, the analysis identifies a dose-response gradient with increasing odds of elevated smallpox rates in local government areas with (i) medium (odds ratio [OR] = 5.32, 95% Confidence Interval [95% CI] 1.96-14.41) and high (OR = 11.32, 95% CI 4.20-31.59) coal mining occupation rates and (ii) medium (OR = 16.74, 95% CI 2.24-125.21) and high (OR = 63.43, 95% CI 7.82-497.21) levels of residential density. The results imply that the spatial transmission of variola virus was facilitated by the close spatial packing of individuals, with a heightened transmission risk in coal mining areas of the country. A syndemic interaction between common respiratory conditions arising from exposure to coal dust and smallpox virus transmission is postulated to have contributed to the findings. We suggest that further studies of the geographical intersection of coal mining and acute infections that are transmitted via respiratory secretions are warranted.

Historical Perspective: What Constitutes Discovery (of a New Virus)?

A historic review of the discovery of new viruses leads to reminders of traditions that have evolved over 118 years. One such tradition gives credit for the discovery of a virus to the investigator(s) who not only carried out the seminal experiments but also correctly interpreted the findings (within the technological context of the day). Early on, ultrafiltration played a unique role in "proving" that an infectious agent was a virus, as did a failure to find any microscopically visible agent, failure to show replication of the agent in the absence of viable cells, thermolability of the agent, and demonstration of a specific immune response to the agent so as to rule out duplicates and close variants. More difficult was "proving" that the new virus was the etiologic agent of the disease ("proof of causation")-for good reasons this matter has been revisited several times over the years as technologies and perspectives have changed. One tradition is that the discoverers get to name their discovery, their new virus (unless some grievous convention has been broken)-the stability of these virus names has been a way to honor the discoverer(s) over the long term. Several vignettes have been chosen to illustrate several difficulties in holding to the traditions (vignettes chosen include vaccinia and variola viruses, yellow fever virus, and influenza viruses. Crimean-Congo hemorrhagic fever virus, Murray Valley encephalitis virus, human immunodeficiency virus 1, Sin Nombre virus, and Ebola virus). Each suggests lessons for the future. One way to assure that discoveries are forever linked with discoverers would be a permanent archive in one of the universal virus databases that have been constructed for other purposes. However, no current database seems ideal-perhaps members of the global community of virologists will have an ideal solution.

Susceptibility of Marmosets (Callithrix jacchus) to Monkeypox Virus: A Low Dose Prospective Model for Monkeypox and Smallpox Disease.

Although current nonhuman primate models of monkeypox and smallpox diseases provide some insight into disease pathogenesis, they require a high titer inoculum, use an unnatural route of infection, and/or do not accurately represent the entire disease course. This is a concern when developing smallpox and/or monkeypox countermeasures or trying to understand host pathogen relationships. In our studies, we altered half of the test system by using a New World nonhuman primate host, the common marmoset. Based on dose finding studies, we found that marmosets are susceptible to monkeypox virus infection, produce a high viremia, and have pathological features consistent with smallpox and monkeypox in humans. The low dose (48 plaque forming units) required to elicit a uniformly lethal disease and the extended incubation (preclinical signs) are unique features among nonhuman primate models utilizing monkeypox virus. The uniform lethality, hemorrhagic rash, high viremia, decrease in platelets, pathology, and abbreviated acute phase are reflective of early-type hemorrhagic smallpox.

Simple technique for in field samples collection in the cases of skin rash illness and subsequent PCR detection of orthopoxviruses and varicella zoster virus.

BACKGROUND: In case of outbreak of rash illness in remote areas, clinically discriminating monkeypox (MPX) from severe form of chickenpox and from smallpox remains a concern for first responders. OBJECTIVE: The goal of the study was therefore to use MPX and chickenpox outbreaks in Democratic Republic of Congo (DRC) as a test case for establishing a rapid and specific diagnosis in affected remote areas. METHODS: In 2008 and 2009, successive outbreaks of presumed MPX skin rash were reported in Bena Tshiadi, Yangala and Ndesha healthcare districts of the West Kasai province (DRC). Specimens consisting of liquid vesicle dried on filter papers or crusted scabs from healing patients were sampled by first responders. A field analytical facility was deployed nearby in order to carry out a real-time PCR (qPCR) assay using genus consensus primers, consensus orthopoxvirus (OPV) and smallpox-specific probes spanning over the 14 kD fusion protein encoding gene. A PCR-restriction fragment length polymorphism was used on-site as backup method to confirm the presence of monkeypox virus (MPXV) in samples. To complete the differential diagnosis of skin rash, chickenpox was tested in parallel using a commercial qPCR assay. In a post-deployment step, a MPXV-specific pyrosequencing was carried out on all biotinylated amplicons generated on-site in order to confirm the on-site results. RESULTS: Whereas MPXV proved to be the agent causing the rash illness outbreak in the Bena Tshiadi, VZV was the causative agent of the disease in Yangala and Ndesha districts. In addition, each on-site result was later confirmed by MPXV-specific pyrosequencing analysis without any discrepancy. CONCLUSION: This experience of rapid on-site dual use DNA-based differential diagnosis of rash illnesses demonstrates the potential of combining tests specifically identifying bioterrorism agents and agents causing natural outbreaks. This opens the way to rapid on-site DNA-based identification of a broad spectrum of causative agents in remote areas.

Poxvirus viability and signatures in historical relics.

Although it has been >30 years since the eradication of smallpox, the unearthing of well-preserved tissue material in which the virus may reside has called into question the viability of variola virus decades or centuries after its original occurrence. Experimental data to address the long-term stability and viability of the virus are limited. There are several instances of well-preserved corpses and tissues that have been examined for poxvirus viability and viral DNA. These historical specimens cause concern for potential exposures, and each situation should be approached cautiously and independently with the available information. Nevertheless, these specimens provide information on the history of a major disease and vaccination against it.

An increasing danger of zoonotic orthopoxvirus infections.

On May 8, 1980, the World Health Assembly at its 33(rd) session solemnly declared that the world and all its peoples had won freedom from smallpox and recommended ceasing the vaccination of the population against smallpox. Currently, a larger part of the world population has no immunity not only against smallpox but also against other zoonotic orthopoxvirus infections. Recently, recorded outbreaks of orthopoxvirus diseases not only of domestic animals but also of humans have become more frequent. All this indicates a new situation in the ecology and evolution of zoonotic orthopoxviruses. Analysis of state-of-the-art data on the phylogenetic relationships, ecology, and host range of orthopoxviruses--etiological agents of smallpox (variola virus, VARV), monkeypox (MPXV), cowpox (CPXV), vaccinia (VACV), and camelpox (CMLV)--as well as the patterns of their evolution suggests that a VARV-like virus could emerge in the course of natural evolution of modern zoonotic orthopoxviruses. Thus, there is an insistent need for organization of the international control over the outbreaks of zoonotic orthopoxvirus infections in various countries to provide a rapid response and prevent them from developing into epidemics.

Efficacy of tecovirimat (ST-246) in nonhuman primates infected with variola virus (Smallpox).

Naturally occurring smallpox has been eradicated but remains a considerable threat as a biowarfare/bioterrorist weapon (F. Fleck, Bull. World Health Organ. 81:917-918, 2003). While effective, the smallpox vaccine is currently not recommended for routine use in the general public due to safety concerns (http://www.bt.cdc.gov/agent/smallpox/vaccination). Safe and effective countermeasures, particularly those effective after exposure to smallpox, are needed. Currently, SIGA Technologies is developing the small-molecule oral drug, tecovirimat (previously known as ST-246), as a postexposure therapeutic treatment of orthopoxvirus disease, including smallpox. Tecovirimat has been shown to be efficacious in preventing lethal orthopoxviral disease in numerous animal models (G. Yang, D. C. Pevear, M. H. Davies, M. S. Collett, T. Bailey, et al., J. Virol. 79:13139-13149, 2005; D. C. Quenelle, R. M. Buller, S. Parker, K. A. Keith, D. E. Hruby, et al., Antimicrob. Agents Chemother., 51:689-695, 2007; E. Sbrana, R. Jordan, D. E. Hruby, R. I. Mateo, S. Y. Xiao, et al., Am. J. Trop. Med. Hyg. 76:768-773, 2007). Furthermore, in clinical trials thus far, the drug appears to be safe, with a good pharmacokinetic profile. In this study, the efficacy of tecovirimat was evaluated in both a prelesional and postlesional setting in nonhuman primates challenged intravenously with 1 × 10(8) PFU of Variola virus (VARV; the causative agent of smallpox), a model for smallpox disease in humans. Following challenge, 50% of placebo-treated controls succumbed to infection, while all tecovirimat-treated animals survived regardless of whether treatment was started at 2 or 4 days postinfection. In addition, tecovirimat treatment resulted in dramatic reductions in dermal lesion counts, oropharyngeal virus shedding, and viral DNA circulating in the blood. Although clinical disease was evident in tecovirimat-treated animals, it was generally very mild and appeared to resolve earlier than in placebo-treated controls that survived infection. Tecovirimat appears to be an effective smallpox therapeutic in nonhuman primates, suggesting that it is reasonably likely to provide therapeutic benefit in smallpox-infected humans.

Smallpox: can we still learn from the journey to eradication?

One of the most celebrated achievements of immunology and modern medicine is the eradication of the dreaded plague smallpox. From the introduction of smallpox vaccination by Edward Jenner, to its popularization by Louis Pasteur, to the eradication effort led by Donald Henderson, this story has many lessons for us today, including the characteristics of the disease and vaccine that permitted its eradication, and the obviousness of the vaccine as a vector for other intractable Infectious diseases. The disease itself, interpreted in the light of modern molecular immunology, is an obvious immunopathological disease, which occurs after a latent interval of 1-2 weeks, and manifests as a systemic cell-mediated delayed type hypersensitivity (DTH) syndrome. The vaccine that slayed this dragon was given the name vaccinia, and was thought to have evolved from cowpox virus, but is now known to be most closely related to a poxvirus isolated from a horse. Of interest is the fact that of the various isolates of orthopox viruses, only variola, vaccinia and monkeypox viruses can infect humans. In contrast to the systemic disease of variola, vaccinia only replicates locally at the site of inoculation, and causes a localized DTH response that usually peaks after 7-10 days. This difference in the pathogenicity of variola vs. vaccinia is thought to be due to the capacity of variola to circumvent innate immunity, which allows it to disseminate widely before the adaptive immune response occurs. Thus, the fact that vaccinia virus is attenuated compared to variola, but is still replication competent, makes for its remarkable efficacy as a vaccine, as the localized infection activates all of the cells and molecules of both innate and adaptive immunity. Accordingly vaccinia itself, and not modified replication incompetent vaccina, is the hope for use as a vector in the eradication of additional pathogenic microbes from the globe.

Poxvirus targeting of E3 ligase ß-TrCP by molecular mimicry: a mechanism to inhibit NF-κB activation and promote immune evasion and virulence.

The transcription factor NF-κB is essential for immune responses against pathogens and its activation requires the phosphorylation, ubiquitination and proteasomal degradation of IκBα. Here we describe an inhibitor of NF-κB from vaccinia virus that has a closely related counterpart in variola virus, the cause of smallpox, and mechanistic similarity with the HIV protein Vpu. Protein A49 blocks NF-κB activation by molecular mimicry and contains a motif conserved in IκBα which, in IκBα, is phosphorylated by IKKß causing ubiquitination and degradation. Like IκBα, A49 binds the E3 ligase ß-TrCP, thereby preventing ubiquitination and degradation of IκBα. Consequently, A49 stabilised phosphorylated IκBα (p-IκBα) and its interaction with p65, so preventing p65 nuclear translocation. Serine-to-alanine mutagenesis within the IκBα-like motif of A49 abolished ß-TrCP binding, stabilisation of p-IκBα and inhibition of NF-κB activation. Remarkably, despite encoding nine other inhibitors of NF-κB, a VACV lacking A49 showed reduced virulence in vivo.

Progression of pathogenic events in cynomolgus macaques infected with variola virus.

Smallpox, caused by variola virus (VARV), is a devastating human disease that affected millions worldwide until the virus was eradicated in the 1970 s. Subsequent cessation of vaccination has resulted in an immunologically naive human population that would be at risk should VARV be used as an agent of bioterrorism. The development of antivirals and improved vaccines to counter this threat would be facilitated by the development of animal models using authentic VARV. Towards this end, cynomolgus macaques were identified as adequate hosts for VARV, developing ordinary or hemorrhagic smallpox in a dose-dependent fashion. To further refine this model, we performed a serial sampling study on macaques exposed to doses of VARV strain Harper calibrated to induce ordinary or hemorrhagic disease. Several key differences were noted between these models. In the ordinary smallpox model, lymphoid and myeloid hyperplasias were consistently found whereas lymphocytolysis and hematopoietic necrosis developed in hemorrhagic smallpox. Viral antigen accumulation, as assessed immunohistochemically, was mild and transient in the ordinary smallpox model. In contrast, in the hemorrhagic model antigen distribution was widespread and included tissues and cells not involved in the ordinary model. Hemorrhagic smallpox developed only in the presence of secondary bacterial infections - an observation also commonly noted in historical reports of human smallpox. Together, our results support the macaque model as an excellent surrogate for human smallpox in terms of disease onset, acute disease course, and gross and histopathological lesions.