Cynthia A. Rossi

Efficacy of a human anthrax vaccine in guinea pigs, rabbits, and rhesus macaques against challenge by Bacillus anthracis isolates of diverse geographical origin

The efficacy of a licensed human anthrax vaccine (Anthrax Vaccine Adsorbed (AVA)) was tested in guinea pigs, rabbits, and rhesus macaques against spore challenge by Bacillus anthracis isolates of diverse geographical origin. Initially, groups of Hartley guinea pigs were vaccinated at 0 and 4 weeks with AVA, then challenged intramuscularly at 10 weeks with spores from 33 isolates of B. anthracis. Survival among the vaccinated groups varied from 6 to 100%, although there were no differences in mean time to death among the groups. There was no correlation between isolate virulence and variable number tandem repeat category or protective antigen genotype identified. New Zealand white rabbits were then vaccinated with AVA at 0 and 4 weeks, and challenged at 10 weeks by aerosol with spores from six of the isolates that were highly virulent in vaccinated guinea pigs. AVA completely protected the rabbits from four of the isolates, and protected 90% of the animals from the other two isolates. Subsequently, two of these six isolates were then used to challenge rhesus macaques, previously vaccinated with AVA at 0 and 4 weeks, and challenged at 10 weeks by aerosol. AVA protected 80 and 100% of the animals from these two isolates. These studies demonstrated that, although AVA confers variable protection against different B. anthracis isolates in guinea pigs, it is highly protective against these same isolates in both rabbits and rhesus macaques.

Comparison of individual and combination DNA vaccines for B. anthracis, Ebola virus, Marburg virus and Venezuelan equine encephalitis virus.

Multiagent DNA vaccines for highly pathogenic organisms offer an attractive approach for preventing naturally occurring or deliberately introduced diseases. Few animal studies have compared the feasibility of combining unrelated gene vaccines. Here, we demonstrate that DNA vaccines to four dissimilar pathogens that are known biowarfare agents, Bacillus anthracis, Ebola (EBOV), Marburg (MARV), and Venezuelan equine encephalitis virus (VEEV), can elicit protective immunity in relevant animal models. In addition, a combination of all four vaccines is shown to be equally as effective as the individual vaccines for eliciting immune responses in a single animal species. These results demonstrate for the first time the potential of combined DNA vaccines for these agents and point to a possible method of rapid development of multiagent vaccines for disparate pathogens such as those that might be encountered in a biological attack.

Undiagnosed acute viral febrile illnesses, Sierra Leone.

Various arthropod-borne and hemorrhagic fever viruses should be considered when Lassa fever is suspected.

Nomenclature- and database-compatible names for the two Ebola virus variants that emerged in Guinea and the Democratic Republic of the Congo in 2014.

In 2014, Ebola virus (EBOV) was identified as the etiological agent of a large and still expanding outbreak of Ebola virus disease (EVD) in West Africa and a much more confined EVD outbreak in Middle Africa. Epidemiological and evolutionary analyses confirmed that all cases of both outbreaks are connected to a single introduction each of EBOV into human populations and that both outbreaks are not directly connected. Coding-complete genomic sequence analyses of isolates revealed that the two outbreaks were caused by two novel EBOV variants, and initial clinical observations suggest that neither of them should be considered strains. Here we present consensus decisions on naming for both variants (West Africa: “Makona”, Middle Africa: “Lomela”) and provide database-compatible full, shortened, and abbreviated names that are in line with recently established filovirus sub-species nomenclatures.

Antigenic and molecular characterization of hantavirus isolates from China

Hemorrhagic fever with renal syndrome (HFRS) is caused by certain viruses in the genus Hantavirus, family Bunyaviridae, and is a major public health problem in China. By using molecular and serological tests, we characterized 15 hantaviruses isolated either from patients with HFRS or from rodents captured in endemic areas of China. By cross plaque-reduction neutralization tests performed with rabbit immune sera, we identified two serologically distinct groups of viruses, comprised of those related to Hantaan virus, and those related to Seoul virus. To study the genetic relationships among these viruses, we amplified a 330 base pair region of the medium (M) genome segment of each isolate by reverse transcription and polymerase chain reaction (PCR) and compared the nucleotide sequences to those of other, well-characterized hantaviruses. In addition, we PCR-amplified and analyzed the entire coding region of the small (S) genome segment of each isolate by restriction enzyme digestion with a battery of enzymes. The results of our genetic analyses of both the M and S segments of these isolates confirmed our serological data, indicating that Hantaan and Seoul viruses co-circulate in endemic disease regions of China. We constructed a phylogenetic tree based on multiple alignment of the partial M segment sequences. The resulting dendrogram distinguished three genetic subtypes of Hantaan viruses and one type of Seoul virus.

Rapid Identification of Vector-Borne Flaviviruses by Mass Spectrometry

Flaviviruses are a highly diverse group of RNA viruses classified within the genus Flavivirus, family Flaviviridae. Most flaviviruses are arthropod-borne, requiring a mosquito or tick vector. Several flaviviruses are highly pathogenic to humans; however, their high genetic diversity and immunological relatedness makes them extremely challenging to diagnose. In this study, we developed and evaluated a broad-range Flavivirus assay designed to detect both tick- and mosquito-borne flaviviruses by using RT-PCR/electrospray ionization mass spectrometry (RT-PCR/ESI-MS) on the Ibis T5000 platform. The assay was evaluated with a panel of 13 different flaviviruses. All samples were correctly identified to the species level. To determine the limit of detection for the mosquito-borne primer sets, serial dilutions of RNA from West Nile virus (WNV) were assayed and could be detected down to an equivalent viral titer of 0.2 plaque-forming units/mL. Analysis of flaviviruses in their natural biological background included testing Aedes aegypti mosquitoes that were laboratory-infected with dengue-1 virus. The assay accurately identified the virus within infected mosquitoes, and we determined the average viral genome per mosquito to be 2.0 x 10(6). Using human blood, serum, and urine spiked with WNV and mouse blood and brain tissues from Karshi virus-infected mice, we showed that these clinical matrices did not inhibit the detection of these viruses. Finally, we used the assay to test field-collected Ixodes scapularis ticks collected from sites in New York and Connecticut. We found 16/322 (5% infection rate) ticks positive for deer tick virus, a subtype of Powassan virus. In summary, we developed a single high-throughput Flavivirus assay that could detect multiple tick- and mosquito-borne flaviviruses and thus provides a new analytical tool for their medical diagnosis and epidemiological surveillance.

Ebola Virus Genome Plasticity as a Marker of Its Passaging History: A Comparison of In Vitro Passaging to Non-Human Primate Infection

To identify polymorphic sites that could be used as biomarkers of Ebola virus passage history, we repeatedly amplified Ebola virus (Kikwit variant) in vitro and in vivo and performed deep sequencing analysis of the complete genomes of the viral subpopulations. We then determined the sites undergoing selection during passage in Vero E6 cells. Four locations within the Ebola virus Kikwit genome were identified that together segregate cell culture-passaged virus and virus obtained from infected non-human primates. Three of the identified sites are located within the glycoprotein gene (GP) sequence: the poly-U (RNA editing) site at position 6925, as well as positions 6677, and 6179. One site was found in the VP24 gene at position 10833. In all cases, in vitro and in vivo, both populations (majority and minority variants) were maintained in the viral swarm, with rapid selections occurring after a few passages or infections. This analysis approach will be useful to differentiate whether filovirus stocks with unknown history have been passaged in cell culture and may support filovirus stock standardization for medical countermeasure development.

Crimean-Congo Hemorrhagic Fever, Afghanistan, 2009

In response to an outbreak of Crimean-Congo hemorrhagic fever in western Afghanistan, we measured immunoglobulin G seroprevalence among household members and their animals. Seroprevalence was 11.2% and 75.0% in humans (n = 330) and livestock (n = 132), respectively. Persons with frequent exposure to cattle had an elevated risk of being immunoglobulin G positive.

Identification of a surrogate marker for infection in the African green monkey model of inhalation anthrax.

ABSTRACT In 2001, a bioterrorism attack involving Bacillus anthracis spore-laced letters resulted in 22 cases of inhalation anthrax, with five fatalities. This incident identified gaps in our health care system and precipitated a renewed interest in identifying both therapeutics and rapid diagnostic assays. To address those gaps, well-characterized animal models that resemble the human disease are needed. In addition, a rapid assay for a reliable diagnostic marker is key to the success of these efforts. In this study, we exposed African green monkeys to B. anthracis spores; examined clinical signs and physiological parameters, including fever, heart rate, complete blood count, and bacteremia; and evaluated the PCR assay and electrochemiluminescence (ECL) immunoassay for the biomarkers protective antigen and capsule. The results demonstrated that although there were neither objective clinical nor physiological signs that consistently identified either infection or the onset of clinical anthrax disease, the African green monkey is a suitable animal model exhibiting a disease course similar to that observed in the rhesus model and humans. We also demonstrated that detection of the biomarkers protective antigen and capsule correlated with bacterial loads in the blood of these nonhuman primates. The ECL immunoassay described here is simple and sensitive enough to provide results in one to two hours, making this assay a viable option for use in the diagnosis of anthrax, leading to timely initiation of treatment, which is a key component of B. anthracis therapeutic development.

Seroprevalance of Crimean–Congo haemorrhagic fever in Bulgarian livestock

Crimean–Congo haemorrhagic fever (CCHF) is a tick-borne zoonotic disease. Over the past decade, CCHF cases in humans have emerged in Turkey and reemerged in the Balkan countries, Ukraine and Tajikistan. Occupational contact with infected livestock has been recognized as a common cause of the disease. A cross-sectional seroprevalence study in livestock was conducted in farming communities of an endemic area in Bulgaria, southeastern Europe. Overall, 72% of the tested animals were positive for IgG antibodies to CCHF virus. By the time the animals were one-year old almost 50% had serologic evidence of CCHF infection, and by two years already 80% of them had been infected. The data obtained in this study reflect current situation of CCHF virus infection among livestock in Bulgaria. The results showed active CCHF virus circulation that poses risk for humans to be infected during contacts with animals and requires public health awareness.