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Zika virus (ZIKV) infection is associated with congenital defects and pregnancy loss. Here, we found that 26% of nonhuman primates infected with Asian/American ZIKV in early gestation experienced fetal demise later in pregnancy despite showing few clinical signs of infection. Pregnancy loss due to asymptomatic ZIKV infection may therefore be a common but under-recognized adverse outcome related to maternal ZIKV infection.

Zika virus (ZIKV) infection during pregnancy causes severe developmental defects in newborns, termed congenital Zika syndrome (CZS). Factors contributing to a surge in ZIKV-associated CZS are poorly understood. One possibility is that ZIKV may exploit the antibody-dependent enhancement of infection mechanism, mediated by cross-reactive antibodies from prior dengue virus (DENV) infection, which may exacerbate ZIKV infection during pregnancy. In this study, we investigated the impact of prior DENV infection or no DENV infection on ZIKV pathogenesis during pregnancy in a total of four female common marmosets with five or six fetuses per group. The results showed that negative-sense viral RNA copies increased in the placental and fetal tissues of DENV-immune dams but not in DENV-naïve dams. In addition, viral proteins were prevalent in endothelial cells, macrophages, and neonatal Fc receptor–expressing cells in the placental trabeculae and in neuronal cells in the brains of fetuses from DENV-immune dams. DENV-immune marmosets maintained high titers of cross-reactive ZIKV-binding antibodies that were poorly neutralizing, raising the possibility that these antibodies might be involved in the exacerbation of ZIKV infection. These findings need to be verified in a larger study, and the mechanism involved in the exacerbation of ZIKV infection in DENV-immune marmosets needs further investigation. However, the results suggest a potential negative impact of preexisting DENV immunity on subsequent ZIKV infection during pregnancy in vivo.

Viral hemorrhagic fevers are a group of disease syndromes caused by infection with certain RNA viruses. The disease is marked by a febrile response, malaise, coagulopathy and vascular permeability culminating in death. Case fatality rates can reach 90% depending on the etiologic agent. Currently, there is no approved antiviral treatment. Because of the high case fatality, risk of importation and the potential to use these agents as biological weapons, development of countermeasures to these agents is a high priority. The sporadic nature of disease outbreaks and the ethical issues associated with conducting a human trial for such diseases make human studies impractical; therefore, development of countermeasures must occur in relevant animal models. Non-human primates are superior models to study infectious disease because their immune system is similar to humans and they are good predictors of efficacy in vaccine development and other intervention strategies. This review article summarizes viral hemorrhagic fever non-human primate models.

The National Select Agents Registry Program regulates the possession, use, and transfer of biological agents and toxins that have been determined to have the potential to pose a severe threat to public safety, to animal or plant health, or to animal or plant products. The program requires that an accurate and current inventory be maintained for each select agent held in long-term storage and for each select toxin. Paper-based systems are easy to implement but can be difficult to maintain, particularly as the number of samples that needs to be documented increases. This article chronicles the development of an electronic system that helps facilitate compliance with the select agents program, while maintaining usability for scientists—particularly while operating in high-containment environments. The system records all required details of samples, including characteristics and manipulations associated with a sample, and additional fields can be added should the regulations change. Each sample is given a unique identifier and labels can be printed with barcodes; searching for sample details using a barcode is highly efficient and limits the need for typing when operating in high containment, which is desirable. Users are given unique identifiers and all transactions are associated with each user. The system can accommodate “audit track” freezer hardware to permit linking of each freezer-access with manipulations performed by a particular user. The system is browser-based and communication between the browser and the server is encrypted. For increased security, separate databases can be installed with different log-in requirements.

The National Select Agents Registry Program regulates the possession, use, and transfer of biological agents and toxins that have been determined to have the potential to pose a severe threat to public safety, to animal or plant health, or to animal or plant products. The program requires that an accurate and current inventory be maintained for each select agent held in long-term storage and for each select toxin. Paper-based systems are easy to implement but can be difficult to maintain, particularly as the number of samples that needs to be documented increases. This article chronicles the development of an electronic system that helps facilitate compliance with the select agents program, while maintaining usability for scientists—particularly while operating in high-containment environments. The system records all required details of samples, including characteristics and manipulations associated with a sample, and additional fields can be added should the regulations change. Each sample is given a unique identifier and labels can be printed with barcodes; searching for sample details using a barcode is highly efficient and limits the need for typing when operating in high containment, which is desirable. Users are given unique identifiers and all transactions are associated with each user. The system can accommodate “audit track” freezer hardware to permit linking of each freezer-access with manipulations performed by a particular user. The system is browser-based and communication between the browser and the server is encrypted. For increased security, separate databases can be installed with different log-in requirements.

ABSTRACTSeveral strategies that target anthrax toxin are being developed as therapies for infection by Bacillus anthracis. Although the action of the tripartite anthrax toxin has been extensively studied in vitro, relatively little is known about the presence of toxins during an infection in vivo. We developed a series of sensitive sandwich enzyme-linked immunosorbent assays (ELISAs) for detection of both the protective antigen (PA) and lethal factor (LF) components of the anthrax exotoxin in serum. The assays utilize as capture agents an engineered high-affinity antibody to PA, a soluble form of the extracellular domain of the anthrax toxin receptor (ANTXR2/CMG2), or PA itself. Sandwich immunoassays were used to detect and quantify PA and LF in animals infected with the Ames or Vollum strains of anthrax spores. PA and LF were detected before and after signs of toxemia were observed, with increasing levels reported in the late stages of the infection. These results represent the detection of free PA and LF by ELISA in the systemic circulation of two animal models exposed to either of the two fully virulent strains of anthrax. Simple anthrax toxin detection ELISAs could prove useful in the evaluation of potential therapies and possibly as a clinical diagnostic to complement other strategies for the rapid identification of B. anthracisinfection.

ABSTRACTPassive immunization has been successfully employed for protection against bacterial and viral infections for over 100 years. Immunoglobulin Fc regions play a critical role in the clearance of bacterial pathogens by mediating antibody-dependent and complement-dependent cytotoxicity. Here we show that antibody fragments engineered to recognize the protective antigen component of the B. anthracisexotoxin with high affinity and conjugated to polyethylene glycol (PEG) for prolonged circulation half-life confer significant protection against inhalation anthrax despite their lack of Fc regions. The speed and lower manufacturing cost of bacterially expressed PEGylated antibody fragments could provide decisive advantages for anthrax prophylaxis. Importantly, our results suggest that PEGylated antibody fragments may represent a unique approach for mounting a rapid therapeutic response to emerging pathogen infections.

Passive immunization has been successfully employed for protection against bacterial and viral infections for over 100 years. Immunoglobulin Fc regions play a critical role in the clearance of bacterial pathogens by mediating antibody-dependent and complement-dependent cytotoxicity. Here we show that antibody fragments engineered to recognize the protective antigen component of the B. anthracis exotoxin with high affinity and conjugated to polyethylene glycol (PEG) for prolonged circulation half-life confer significant protection against inhalation anthrax despite their lack of Fc regions. The speed and lower manufacturing cost of bacterially expressed PEGylated antibody fragments could provide decisive advantages for anthrax prophylaxis. Importantly, our results suggest that PEGylated antibody fragments may represent a unique approach for mounting a rapid therapeutic response to emerging pathogen infections.

In this study, Leishmania RNA virus 1-4 (LRV1-4) particles purified from host Leishmania guyanensis promastigotes were examined for capsid endoribonuclease. Temperature optimum for the endoribonulease activity was found to be at 37degrees C to 42degrees C and the activity was specifically inhibited by the aminoglycoside antibiotics, neomycin, kanamycin, and hygromycin and by 100 mM levels of NaCl or KCl. To determine the catalytic domain of the capsid endoribonuclease activity, three point-mutation at cysteine residues at C47S (P1), C128/ 133S (P2), and C194R (P3) were prepared and each gene was constructed into baculoviruses and expressed in Sf9 insect cells. LRV1-4 capsid N- terminus (N2 and N3) and C-terminus (C1 and C2) deletion mutants (Cadd et al., 1994) were also examined by in vitro RNA cleavage assay. The results showed that the capsid mutants; C1, C2, N3, P1, and P2 were capable of forming proper virus-like particles (VLPs) and they all possessed the specific endoribonuclease activity. However, two assembly-defective capsid mutants, N2 (N- terminus 24-amino acids deletion) and P3 mutants, did not retain the specific endoribonuclease activity. Taken together, the results suggest that at least 24 amino acids from the N-terminal region and C194 residue in LRV1-4 capsid protein are functionally important for LRV1-4 viral assembly and the capsid endoribonuclease activity may be dependent upon the properly assembled LRV1-4 virus particles.

ABSTRACTThe LeishmaniaRNA virus 1-4 capsid protein possesses an endoribonuclease activity responsible for single-site-specific cleavage within the 450-nucleotide 5' untranslated region of its own viral RNA transcript. To characterize the minimal essential RNA determinants required for site-specific cleavage, mutated RNA transcripts were examined for susceptibility to cleavage by the virus capsid protein in an in vitro assay. Deletion analyses revealed that all determinants necessary for accurate cleavage are encoded in viral nucleotides 249 to 342. Nuclease mapping and site-specific mutagenesis of the minimal RNA sequence defined a stem-loop structure that is located 40 nucleotides upstream from the cleavage site (nucleotide 320) and that is essential for accurate RNA cleavage. Abrogation of cleavage by disruption of base pairing within the stem-loop was reversed through the introduction of complementary nucleotide substitutions that reestablished the structure. We also provide evidence that divalent cations, essential components of the cleavage reaction, stabilized the stem-loop structure in solution. That capsid-specific antiserum eliminated specific RNA cleavage provides further evidence that the virus capsid gene encodes the essential endoribonuclease activity.

A protein which produces a thermal hysteresis (a difference between the freezing and melting points) was purified from the hemolymph of the milkweed bug,Oncopeltus fasciatus. The amino acid composition of theOncopeltus thermal hysteresis protein is somewhat different from that of the larvae of the beetle,Tenebrio molitor, which is the only other insect from which such a protein has as yet been purified. The major difference between the two is the large amount of serine (30.5% of the amino acid residues) and glycine (20.0%) present in theO. fasciatus protein. Both insect proteins have a composition which consists of approximately 60% polar amino acids and lacks large amounts of alanine. In these respects they are quite different from the fish protein antifreezes. The apparent differences in structure of the thermal hysteresis proteins and glycoproteins indicates that these proteins have evolved independently and therefore offer an interesting example of convergent evolution.

Certain strains of Leishmania guyanensis carry persistently infecting double-stranded RNA viruses. The viral polymerase has been shown to have both transcriptase and replicase activity. To date, only full-length RNA transcription of minus and plus strands have been reported. This report describes the synthesis of a 320 nt transcript which is complementary to the 3' end of the minus strand RNA. This plus-stranded short transcript was first detected in an in vitro polymerase assay. It was also detected in virus-infected Leishmania cells by a reverse transcription-polymerase chain reaction assay and by Northern analysis of infected Leishmania cell RNA. Copyright 1994, 1999 Academic Press

In order to understand the coding strategies and identify potential cis-acting sequences in Leishmania RNA virus 1 (LRV1), a complete cDNA sequence was obtained for LRV1-4 and compared to the sequence reported for LRV1-1. The results show that the 5' end of LRV1 is conserved at the nucleotide level while open reading frames (ORFs) 2 and 3 are conserved at the amino acid level. A simple translation initiation consensus sequence is conserved at the 5' end of ORF2 but absent from ORF3, consistent with a possibility that ORF3 is expressed as a gag-pol fusion protein. Comparison of secondary structure predictions obtained for both isolates identified nucleotide sequences capable of forming conserved stem-loops at the virus termini and in the putative frameshift region between ORF2 and ORF3. Although direct evidence is lacking, the appearance of compensatory nucleotide substitutions suggests that the structures may form in vivo. Possible functions for the conserved structures are discussed. Copyright 1994, 1999 Academic Press

The haemolymph of larvae of Tenebrio molitor contains a factor which produces a thermal hysteresis (a difference between the freezing and melting points) of approximately 0·75 °C. When larvae were acclimated to low temperatures or short photoperiod the thermal hysteresis increased more than twofold. Coincident with the increase in thermal hysteresis the supercooling points and lower lethal temperatures of the larvae were depressed. Therefore, the thermal-hysteresis-producing factor seems to function as an antifreeze. The factor may also act as an adaptation to prevent desiccation. Thermal hysteresis increased almost three-fold in larvae acclimated to low relative humidity. Also, larvae with high levels of the thermal hysteresis factor survived low relative humidities much better than did larvae with lower levels.

Leishmaniaviruses (LRV) are double-stranded RNA viruses that persistently infect some strains of the protozoan parasite Leishmania. The identification of a short viral RNA transcript led to our discovery of an endoribonuclease activity of the LRV capsid protein. Other known endoribonucleases serve a variety of diverse roles in the regulated balance of processing and degradation of both cellular and viral RNAs, thus determining the amount and functionality of specific RNA molecules in a cell at any given time. The consequence of LRV RNA cleavage on the LRV life cycle has not yet been determined. Here we review the LRV endoribonuclease and discuss potential roles for this endoribonuclease activity in the context of the involvement of other endoribonucleases in regulating viral gene expression and replicative capacity. J. Exp. Zool. 282:254–260, 1998. © 1998 Wiley-Liss, Inc.

The genome of La Crosse virus, a member of the Bunyaviridae, is made up of three molecules. Circular nucleocapsid structures, in three size classes, have been isolated from La Crosse virus (Obijeski et al. J. Virol. 20:664-675, 1976). Recently, Obijeski et al. (Nucleic Acids Res. 8:2431-2438) have found that the 5' and 3' ends of each segment are complementary in sequence. We determined that a 5' and 3' end complementary structure, predicted by the rules of Tinoco et al. (Nature [London] 230:362-367), can and will anneal under certain conditions. This structure is resistant to RNase in high-salt medium and can be isolated in a reasonably high yield.

ABSTRACTAdenoviruses (AdVs) are DNA viruses that infect many vertebrate hosts, including humans and nonhuman primates. Here we identify a novel AdV species, provisionally named “simian adenovirus C (SAdV-C),” associated with a 1997 outbreak of acute respiratory illness in captive baboons (4 of 9) at a primate research facility in Texas. None of the six AdVs recovered from baboons (BaAdVs) during the outbreak, including the two baboons who died from pneumonia, were typeable. Since clinical samples from the two fatal cases were not available, whole-genome sequencing of nasal isolates from one sick baboon and three asymptomatic baboons during the outbreak was performed. Three AdVs were members of species SAdV-C (BaAdV-2 and BaAdV-4 were genetically identical, and BaAdV-3), while one (BaAdV-1) was a member of the recently described SAdV-B species. BaAdV-3 was the only AdV among the 4 isolated from a sick baboon, and thus was deemed to be the cause of the outbreak. Significant divergence (<58% amino acid identity) was found in one of the fiber proteins of BaAdV-3 relative to BaAdV-2 and -4, suggesting that BaAdV-3 may be a rare SAdV-C recombinant. Neutralizing antibodies to the other 3 AdVs, but not BaAdV-3, were detected in healthy baboons from 1996 to 2003 and staff personnel from 1997. These results implicate a novel adenovirus species (SAdV-C) in an acute respiratory outbreak in a baboon colony and underscore the potential for cross-species transmission of AdVs between humans and nonhuman primates.IMPORTANCEAdenoviruses (AdVs) are DNA viruses that infect many animals, including humans and monkeys. In 1997, an outbreak of acute respiratory illness from AdVs occurred in a baboon colony in Texas. Here we use whole-genome sequencing and antibody testing to investigate new AdVs in baboons (BaAdVs) during the outbreak, one of which, BaAdV-3, came from a sick animal. By sequence analysis, BaAdV-3 may be a recombinant strain that arose from a related BaAdV found in baboons nearby in the colony (who were not sick) and yet another unknown AdV. We also found antibodies to these new BaAdVs in baboons and staff personnel at the facility. Taken together, our findings of a new AdV species as the cause of an acute respiratory outbreak in a baboon colony underscore the ongoing threat from emerging viruses that may carry the potential for cross-species transmission between monkeys and humans.

The demand for nonhuman primates will undoubtedly increase to meet biomedical needs in this current age of biodefense. The availability of funding has increased the research on select agents and has created a requirement to validate results in relevant primate models. This review provides a description of current and potential biological threats that are likely to require nonhuman primates for the development of vaccines and therapeutics. Primates have been an invaluable resource in the dissection of viral disease pathogenesis as well as in testing vaccine efficacy. DNA vaccine approaches have been studied successfully for Ebola, Lassa, and anthrax in nonhuman primate models. Nonhuman primate research with monkeypox has provided insight into the role of cytokines in limiting disease severity. Biodefense research that has focused on select agents of bacterial origin has also benefited from nonhuman primate studies. Rhesus macaques have traditionally been the model of choice for anthrax research and have yielded successful findings in vaccine development. In plague research, African green monkeys have contributed to vaccine development. However, the disadvantages of current vaccines will undoubtedly require the generation of new vaccines, thus increasing the need for nonhuman primate research. Unfortunately, the current biosafety level (BSL)-3 and BSL-4 facilities equipped to perform this research are limited, which may ultimately impede progress in this era of biodefense.

Ribavirin (l-β-d-ribofuranosyl-l,2,4-triazole-3-carboxamide) is a broad-spectrum antiviral agent whose molecular mode of action remains remarkably controversial. This antiviral agent was approved by the U.S. Food and Drug Administration in 1986 for use as an aerosol for infants with serious infections due to respiratory syncytial virus. Ribavirin is and has been under clinical investigation for activity against a variety of viral illnesses, including those due to influenza virus, Lassa fever virus, Hantaan virus, and human immunodeficiency virus (HIV). There has been a great deal of clinical interest in the utilization of ribavirin for treatment of infections due to HIV. It has been reported to slow the development of AIDS in HIV-infected patients. We describe here the major mechanisms of action of this newly licensed antiviral agent.

We have examined the effect of ribavirin on vesicular stomatitis virus (VSV) RNA synthesis using a cell-free transcription system. Our prior data indicated that ribavirin had little effect on primary transcription of VSV mRNAs synthesized intracellularly, but that these RNAs were translated inefficiently. Consistent with these data, VSV cell-free transcription was inhibited only mildly (25%) by ribavirin triphosphate at 100 mg/ml. However, ribavirin diphosphate (RDF) and, to a lesser extent, ribavirin monophosphate, were 2-3 times as inhibitory at the same concentration. Transcripts synthesized in the presence of RDP were full lengthed. The inhibition by RDP could be reversed by increasing the concentration of GTP. However, reversal could also be achieved by increasing the concentration of UTP, CTP, and ATP, but not by the addition of GDP. RDP was added to a LaCrosse virus cell-free transcription assay to determine if an inhibitory effect could be established in a viral system more sensitive to ribavirin than VSV; addition of RDP to this reaction led to profound inhibition of RNA synthesis at concentrations as low as 0.1 ug/ml. These data suggest that in some viruses ribavirin may have a major direct effect on initial steps of transcription and that this effect may be mediated by the di- and monophosphorylated forms.