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Concept: Vaccinia

360

Smallpox holds a unique position in the history of medicine. It was the first disease for which a vaccine was developed and remains the only human disease eradicated by vaccination. Although there have been claims of smallpox in Egypt, India, and China dating back millennia [1-4], the timescale of emergence of the causative agent, variola virus (VARV), and how itĀ evolved in the context of increasingly widespread immunization, have proven controversial [4-9]. In particular, some molecular-clock-based studies have suggested that key events in VARV evolution only occurred during the last two centuries [4-6] and hence in apparent conflict with anecdotal historical reports, although it is difficult to distinguish smallpox from other pustular rashes by description alone. To address these issues, we captured, sequenced, and reconstructed a draft genome of an ancient strain of VARV, sampled from a Lithuanian child mummy dating between 1643 and 1665 and close to the time of several documented European epidemics [1, 2, 10]. When compared to vaccinia virus, this archival strain contained the same pattern of gene degradation as 20(th) century VARVs, indicating that such loss of gene function had occurred before ca. 1650. Strikingly, the mummy sequence fell basal to all currently sequenced strains of VARV on phylogenetic trees. Molecular-clock analyses revealed a strong clock-like structure and that the timescale of smallpox evolution is more recent than often supposed, with the diversification of major viral lineages only occurring within the 18(th) and 19(th) centuries, concomitant with the development of modern vaccination.

Concepts: Poxviridae, Vaccine, Cowpox, Vaccinia, Virus, Infectious disease, Smallpox, Vaccination

211

Edward Jenner and his contemporaries believed that his variolae vaccinae originated in horses and molecular analyses show that modern vaccinia virus (VACV) strains share common ancestry with horsepox virus (HPXV). Given concerns relating to the toxicity of modern VACV vaccines, we asked whether an HPXV-based vaccine might provide a superior alternative. Since HPXV may be extinct and the only specimen of HPXV that has been identified is unavailable for investigation, we explored whether HPXV could be obtained by large-scale gene synthesis. Ten large (10-30 kb) fragments of DNA were synthesized based on the HPXV sequence along with two 157 nt VACV terminal sequences, and were recombined into a live synthetic chimeric HPXV (scHPXV) in cells infected with Shope fibroma virus (SFV). Sequencing of the 212 kbp scHPXV confirmed it encoded a faithful copy of the input DNA. We believe this is the first complete synthesis of a poxvirus using synthetic biology approaches. This scHPXV produced smaller plaques, produced less extracellular virus and exhibited less virulence in mice than VACV, but still provided vaccine protection against a lethal VACV challenge. Collectively, these findings support further development of scHPXV as a novel replication-proficient smallpox vaccine.

Concepts: Virus, Gene, Vaccinia, Edward Jenner, Vaccination, Vaccine, Cowpox, Smallpox

169

BACKGROUND: There is agreement that the infectivity assay with the duck hepatitis B virus (DHBV) is a suitable surrogate test to validate disinfectants for hepatitis B virucidal activity. However, since this test is not widely used, information is necessary whether disinfectants with limited virucidal activity also inactivate DHBV. In general, disinfectants with limited virucidal activity are used for skin and sensitive surfaces while agents with full activity are more aggressive. The present study compares the activity of five different biocides against DHBV and the classical test virus for limited virucidal activity, the vaccinia virus strain Lister Elstree (VACV) or the modified vaccinia Ankara strain (MVA). METHODS: Virucidal assay was performed as suspension test according to the German DVV/RKI guideline. Duck hepatitis B virus obtained from congenitally infected Peking ducks was propagated in primary duck embryonic hepatocytes and was detected by indirect immunofluorescent antigen staining. RESULTS: The DHBV was inactivated by the use of 40% ethanol within 1-min and 30% isopropanol within 2-min exposure. In comparison, 40% ethanol within 2-min and 40% isopropanol within 1-min exposure were effective against VACV/MVA. These alcohols only have limited virucidal activity, while the following agents have full activity. 0.01% peracetic acid inactivated DHBV within 2 min and a concentration of 0.005% had virucidal efficacy against VACV/MVA within 1 min. After 2-min exposure, 0.05% glutardialdehyde showed a comparable activity against DHBV and VACV/MVA. This is also the case for 0.7% formaldehyde after a contact time of 30 min. CONCLUSIONS: Duck hepatitis B virus is at least as sensitive to limited virucidal activity as VACV/MVA. Peracetic acid is less effective against DHBV, while the alcohols are less effective against VACV/MVA. It can be expected that in absence of more direct tests the results may be extrapolated to HBV.

Concepts: Ethanol, Hepadnaviridae, Vaccinia, Modified vaccinia Ankara, Alcohol, Viruses, Virus, Hepatitis B

28

Poxviruses are widespread pathogens, which display extremely different host ranges. Whereas some poxviruses, including variola virus, display narrow host ranges, others such as cowpox viruses naturally infect a wide range of mammals. The molecular bases for differences in host range are poorly understood but apparently depend on the successful manipulation of the host antiviral response. Some poxvirus genes have been shown to confer host tropism in experimental settings and are thus called host range factors. Identified host range genes include vaccinia virus K1L, K3L, E3L, B5R, C7L and SPI-1, cowpox virus CP77/CHOhr, ectromelia virus p28 and 022, and myxoma virus T2, T4, T5, 11L, 13L, 062R and 063R. These genes encode for ankyrin repeat-containing proteins, tumor necrosis factor receptor II homologs, apoptosis inhibitor T4-related proteins, Bcl-2-related proteins, pyrin domain-containing proteins, cellular serine protease inhibitors (serpins), short complement-like repeats containing proteins, KilA-N/RING domain-containing proteins, as well as inhibitors of the double-stranded RNA-activated protein kinase PKR. We conducted a systematic survey for the presence of known host range genes and closely related family members in poxvirus genomes, classified them into subgroups based on their phylogenetic relationship and correlated their presence with the poxvirus phylogeny. Common themes in the evolution of poxvirus host range genes are lineage-specific duplications and multiple independent inactivation events. Our analyses yield new insights into the evolution of poxvirus host range genes. Implications of our findings for poxvirus host range and virulence are discussed.

Concepts: Vaccination, Gene, Poxviruses, Cowpox, Vaccinia, Poxviridae, Smallpox, Virus

28

To ensure spread from one cell to another, exocytosed vaccinia virions recruit cellular actin polymerization machinery to blast off from the cell surface on actin tails. Humphries et al. (2012) now show that the virus exploits clathrin to organize viral factors into a launch pad that facilitates efficient actin tail formation.

Concepts: Radio resource management, Gene, Organism, DNA, Endocytosis, Protein, Vaccinia, Virus

26

We assessed several routes of immunization with vaccinia virus (VACV) in protecting against the murine poxvirus, ectromelia (ECTV). By a wide margin, skin scarification provided the greatest protection. Humoral immunity and resident-memory T cells notwithstanding, several approaches revealed that circulating, memory CD8(+) T-cells primed via scarification were functionally superior and conferred enhanced virus control. Immunization via the epithelial route warrants further investigation as it may also provide enhanced defense against other infectious agents.

Concepts: Humoral immunity, Immunology, Virus, Vaccinia, B cell, Smallpox, Adaptive immune system, Immune system

23

We studied toxicity and antiviral activity of bioactive substances extracted from the roots (ethylacetate extracts) and aerial parts (ethanol extracts) of lady’s mantle (Alchemilla vilgaris L.). Plant extracts are characterized by low toxicity for continuous Vero cell culture, but inhibit the reproduction of orthopoxviruses (vaccinia virus and ectromelia virus) in these cells. Of all studied extracts, ethylacetate extract from lady’s mantle roots characterized by the highest content of catechins in comparison with other samples demonstrated the highest activity in vitro towards the studied viruses (neutralization index for vaccinia and ectromelia viruses were 4.0 and 3.5 lg, respectively). The antiviral effect of Alchemilla vulgaris L. extracts was shown to be dose dependent.

Concepts: Antiviral drug, Alchemilla, Ectromelia virus, Vero cell, Vaccinia, Cell lines, Organism, Virus

10

After Edward Jenner established human vaccination over 200 years ago, attenuated poxviruses became key players to contain the deadliest virus of its own family: Variola virus (VARV), the causative agent of smallpox. Cowpox virus (CPXV) and horsepox virus (HSPV) were extensively used to this end, passaged in cattle and humans until the appearance of vaccinia virus (VACV), which was used in the final campaigns aimed to eradicate the disease, an endeavor that was accomplished by the World Health Organization (WHO) in 1980. Ever since, naturally evolved strains used for vaccination were introduced into research laboratories where VACV and other poxviruses with improved safety profiles were generated. Recombinant DNA technology along with the DNA genome features of this virus family allowed the generation of vaccines against heterologous diseases, and the specific insertion and deletion of poxvirus genes generated an even broader spectrum of modified viruses with new properties that increase their immunogenicity and safety profile as vaccine vectors. In this review, we highlight the evolution of poxvirus vaccines, from first generation to the current status, pointing out how different vaccines have emerged and approaches that are being followed up in the development of more rational vaccines against a wide range of diseases.

Concepts: Virus, Poxviridae, Vaccinia, DNA, Edward Jenner, Cowpox, Vaccination, Smallpox

8

On June 25, 2015, the Advisory Committee on Immunization Practices (ACIP) recommended routine vaccination with live smallpox (vaccinia) vaccine (ACAM2000) for laboratory personnel who directly handle 1) cultures or 2) animals contaminated or infected with replication-competent vaccinia virus, recombinant vaccinia viruses derived from replication-competent vaccinia strains (i.e., those that are capable of causing clinical infection and producing infectious virus in humans), or other orthopoxviruses that infect humans (e.g., monkeypox, cowpox, and variola) (recommendation category: A, evidence type 2 [Box]). Health care personnel (e.g., physicians and nurses) who currently treat or anticipate treating patients with vaccinia virus infections and whose contact with replication-competent vaccinia viruses is limited to contaminated materials (e.g., dressings) and persons administering ACAM2000 smallpox vaccine who adhere to appropriate infection prevention measures can be offered vaccination with ACAM2000 (recommendation category: B, evidence type 2 [Box]). These revised recommendations update the previous ACIP recommendations for nonemergency use of vaccinia virus smallpox vaccine for laboratory and health care personnel at risk for occupational exposure to orthopoxviruses (1). Since 2001, when the previous ACIP recommendations were developed, ACAM2000 has replaced Dryvax as the only smallpox vaccine licensed by the U.S. Food and Drug Administration (FDA) and available for use in the United States (2). These recommendations contain information on ACAM2000 and its use in laboratory and health care personnel at risk for occupational exposure to orthopoxviruses.

Concepts: Edward Jenner, Infectious disease, Cowpox, Infection, Vaccination, Vaccinia, Virus, Smallpox

4

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.

Concepts: Vaccine, Biological warfare, Poxviridae, Cowpox, Vaccinia, Vaccination, Infectious disease, Smallpox