Lessons from the pandemic: Responding to emerging zoonotic viral diseases-a Keystone Symposia report.

The COVID-19 pandemic caught the world largely unprepared, including scientific and policy communities. On April 10-13, 2022, researchers across academia, industry, government, and nonprofit organizations met at the Keystone symposium "Lessons from the Pandemic: Responding to Emerging Zoonotic Viral Diseases" to discuss the successes and challenges of the COVID-19 pandemic and what lessons can be applied moving forward. Speakers focused on experiences not only from the COVID-19 pandemic but also from outbreaks of other pathogens, including the Ebola virus, Lassa virus, and Nipah virus. A general consensus was that investments made during the COVID-19 pandemic in infrastructure, collaborations, laboratory and manufacturing capacity, diagnostics, clinical trial networks, and regulatory enhancements-notably, in low-to-middle income countries-must be maintained and strengthened to enable quick, concerted responses to future threats, especially to zoonotic pathogens.

COVID-19 Serology Control Panel Using the Dried-Tube Specimen Method.

The dried-tube specimen (DTS) procedure was used to develop the COVID-19 serology control panel (CSCP). The DTS offers the benefit of shipping materials without a cold chain, allowing for greater access without deterioration of material integrity. Samples in the panel were sourced from COVID-19 convalescent persons from March to May 2020. The immunoglobulin subtypes (total Ig, IgM, and IgG) and their respective reactivity to severe acute respiratory syndrome coronavirus 2 nucleocapsid, spike, and receptor-binding domain antigens of the samples were delineated and compared with the WHO International Standard to elucidate the exact binding antibody units of each CSCP sample and ensure the CSCP provides adequate reactivity for different types of serological test platforms. We distribute the CSCP as a kit with five coded tubes to laboratories around the world to be used to compare test kits for external quality assurance, for harmonizing laboratory testing, and for use as training materials for laboratory workers.

Household Materials Selection for Homemade Cloth Face Coverings and Their Filtration Efficiency Enhancement with Triboelectric Charging.

The COVID-19 pandemic is currently causing a severe disruption and shortage in the global supply chain of necessary personal protective equipment (e.g., N95 respirators). The U.S. CDC has recommended use of household cloth by the general public to make cloth face coverings as a method of source control. We evaluated the filtration properties of natural and synthetic materials using a modified procedure for N95 respirator approval. Common fabrics of cotton, polyester, nylon, and silk had filtration efficiency of 5-25%, polypropylene spunbond had filtration efficiency 6-10%, and paper-based products had filtration efficiency of 10-20%. An advantage of polypropylene spunbond is that it can be simply triboelectrically charged to enhance the filtration efficiency (from 6 to >10%) without any increase in pressure (stable overnight and in humid environments). Using the filtration quality factor, fabric microstructure, and charging ability, we are able to provide an assessment of suggested fabric materials for homemade facial coverings.

Innovative and New Approaches to Laboratory Diagnosis of Zika and Dengue: A Meeting Report.

Epidemics of dengue, Zika, and other arboviral diseases are increasing in frequency and severity. Current efforts to rapidly identify and manage these epidemics are limited by the short diagnostic window in acute infection, the extensive serologic cross-reactivity among flaviviruses, and the lack of point-of-care diagnostic tools to detect these viral species in primary care settings. The Partnership for Dengue Control organized a workshop to review the current landscape of Flavivirus diagnostic tools, identified current gaps, and developed strategies to accelerate the adoption of promising novel technologies into national programs. The rate-limiting step to bringing new diagnostic tools to the market is access to reference materials and well-characterized clinical samples to facilitate performance evaluation. We suggest the creation of an international laboratory-response consortium for flaviviruses with a decentralized biobank of well-characterized samples to facilitate assay validation. Access to proficiency panels are needed to ensure quality control, in additional to in-country capacity building.

The laboratory efficiencies initiative: partnership for building a sustainable national public health laboratory system.

Beginning in early 2011, the Centers for Disease Control and Prevention and the Association of Public Health Laboratories launched the Laboratory Efficiencies Initiative (LEI) to help public health laboratories (PHLs) and the nation's entire PHL system achieve and maintain sustainability to continue to conduct vital services in the face of unprecedented financial and other pressures. The LEI focuses on stimulating substantial gains in laboratories' operating efficiency and cost efficiency through the adoption of proven and promising management practices. In its first year, the LEI generated a strategic plan and a number of resources that PHL directors can use toward achieving LEI goals. Additionally, the first year saw the formation of a dynamic community of practitioners committed to implementing the LEI strategic plan in coordination with state and local public health executives, program officials, foundations, and other key partners.

Global networking for biodosimetry laboratory capacity surge in radiation emergencies.

For the public health management of radiation emergencies, one of the essential components of integrated risk assessment is to quickly and accurately assess and categorize the exposure. In addition to other methods, biodosimetry is instrumental to support decision-making for: 1) efficient secondary triage in a hospital response phase; 2) multi-parameter approach for defining best-treatment strategies for those severely exposed; 3) clinical prognosis and assessment of risk; and 4) reassurance and psychological support for those potentially exposed, or "worried-well." In large-scale events, the number of victims, and especially those worried-well, is likely to overwhelm hospital and laboratory capacities in the accident area. This is already being addressed through the networking approach within several countries and/or regions of the world. The paper reports about WHO's activity toward coordination of these regional efforts and the international collaborative network of biodosimetry laboratory services, WHO BioDoseNet. The network includes more than 30 laboratories around the world and supports the implementation of the revised International Health Regulations, the scope of which since 2007 also covers the field of radionuclear incidents.

Survival of secondary lethal systemic Francisella LVS challenge depends largely on interferon gamma.

Although survival of primary infection with the live vaccine strain (LVS) of Francisella tularensis depends on interferon gamma (IFN-gamma), the relative importance of IFN-gamma to secondary protective immunity in vivo has not been clearly established. Here we examine the role of IFN-gamma in T cell priming and expression of vaccine-induced protection against lethal intraperitoneal challenge of mice. Large amounts of IFN-gamma were detected between days 3 and 7 in the sera of LVS-immunized mice, while relatively small amounts were found transiently after secondary LVS challenge. Consistent with the production of this cytokine, mice lacking IFN-gamma (gamma interferon knockout, GKO, mice) could not be successfully vaccinated with LVS or an attenuated mglA mutant of F. novicida to withstand secondary Francisella LVS challenge. Further, splenocytes from such primed mice did not adoptively transfer protection to naive GKO recipient mice in vivo, nor control the intramacrophage growth of LVS in vitro. Finally, LVS-immune WT mice depleted of IFN-gamma prior to intraperitoneal challenge survived only the lowest doses of challenge. Thus successful priming of protective LVS-immune T cells, as well as complete expression of protection against Francisella during secondary challenge, depends heavily on IFN-gamma.

New approaches to diagnosis and therapy of tularemia.

Francisella tularensis is a potent pathogen and a cause of severe human disease. The outcome of tularemia will depend on rapid insertion of appropriate antibiotics. Until recently, effective clinical handling was hampered by shortcomings in laboratory diagnostics. No suitable direct methods were available and, because of risks and isolate recovery difficulties associated with laboratory work, culture has been rarely practiced. Due to achievements from work on modern technology, however, tularemia can now be rapidly and specifically diagnosed. Conventional PCR has been successfully applied on wound specimens of patients acquiring tularemia, and prospects for application on other human specimens are promising. Besides allowing diagnostics at high sensitivity and specificity, the PCR technology will also facilitate the identification of cases of tularemia presenting with aberrant signs and symptoms. Antibiotics for efficacious treatment of tularemia have been available for several decades. Although highly valuable, these drugs are afflicted with adverse effects and/or are available only for parenteral therapy. Recently, quinolones have been shown to afford a new valuable option for treatment of tularemia caused by F. tularensis subsp. holarctica (type B). Experience in treating more severe disease caused by F. tularensis subsp. tularensis (type A) is currently limited. In essence, the clinical handling of tularemia is currently facilitated by new achievements in molecular diagnostics and, at least with regard to type B tularemia, by the introduction of quinolones for therapy.

Phenotypic and molecular characterizations of Yersinia pestis isolates from Kazakhstan and adjacent regions.

Recent interest in characterizing infectious agents associated with bioterrorism has resulted in the development of effective pathogen genotyping systems, but this information is rarely combined with phenotypic data. Yersinia pestis, the aetiological agent of plague, has been well defined genotypically on local and worldwide scales using multi-locus variable number tandem repeat analysis (MLVA), with emphasis on evolutionary patterns using old isolate collections from countries where Y. pestis has existed the longest. Worldwide MLVA studies are largely based on isolates that have been in long-term laboratory culture and storage, or on field material from parts of the world where Y. pestis has potentially circulated in nature for thousands of years. Diversity in these isolates suggests that they may no longer represent the wild-type organism phenotypically, including the possibility of altered pathogenicity. This study focused on the phenotypic and genotypic properties of 48 Y. pestis isolates collected from 10 plague foci in and bordering Kazakhstan. Phenotypic characterization was based on diagnostic tests typically performed in reference laboratories working with Y. pestis. MLVA was used to define the genotypic relationships between the central-Asian isolates and a group of North American isolates, and to examine Kazakh Y. pestis diversity according to predefined plague foci and on an intermediate geographical scale. Phenotypic properties revealed that a large portion of this collection lacks one or more plasmids necessary to complete the blocked flea/mammal transmission cycle, has lost Congo red binding capabilities (Pgm-), or both. MLVA analysis classified isolates into previously identified biovars, and in some cases groups of isolates collected within the same plague focus formed a clade. Overall, MLVA did not distinguish unique phylogeographical groups of Y. pestis isolates as defined by plague foci and indicated higher genetic diversity among older biovars.

Complete genome sequence of Yersinia pestis strains Antiqua and Nepal516: evidence of gene reduction in an emerging pathogen.

Yersinia pestis, the causative agent of bubonic and pneumonic plagues, has undergone detailed study at the molecular level. To further investigate the genomic diversity among this group and to help characterize lineages of the plague organism that have no sequenced members, we present here the genomes of two isolates of the "classical" antiqua biovar, strains Antiqua and Nepal516. The genomes of Antiqua and Nepal516 are 4.7 Mb and 4.5 Mb and encode 4,138 and 3,956 open reading frames, respectively. Though both strains belong to one of the three classical biovars, they represent separate lineages defined by recent phylogenetic studies. We compare all five currently sequenced Y. pestis genomes and the corresponding features in Yersinia pseudotuberculosis. There are strain-specific rearrangements, insertions, deletions, single nucleotide polymorphisms, and a unique distribution of insertion sequences. We found 453 single nucleotide polymorphisms in protein-coding regions, which were used to assess the evolutionary relationships of these Y. pestis strains. Gene reduction analysis revealed that the gene deletion processes are under selective pressure, and many of the inactivations are probably related to the organism's interaction with its host environment. The results presented here clearly demonstrate the differences between the two biovar antiqua lineages and support the notion that grouping Y. pestis strains based strictly on the classical definition of biovars (predicated upon two biochemical assays) does not accurately reflect the phylogenetic relationships within this species. A comparison of four virulent Y. pestis strains with the human-avirulent strain 91001 provides further insight into the genetic basis of virulence to humans.

Francisella tularensis in rodents, China.

A total of 420 rodents in China were examined for Francisella tularensis by polymerase chain reaction. The infection rates were 4.76% in total, and 11.65%, 10.00%, 6.56%, 1.77%, and 0% in Jilin, Xinjiang, Heilongjiang, Inner Mongolia, and Zhejiang, respectively. Sequence analysis showed that all the detected agents belonged to F. tularensis subsp. holarctica.

Identifying sources of human exposure to plague.

Yersinia pestis, the etiologic agent of plague, has shaped the course of human history, killing millions of people in three major pandemics. This bacterium is still endemic in parts of Asia, Africa, and the Americas, where it poses a natural disease threat to human populations. Y. pestis has also recently received attention as a possible bioterrorism agent. Thus, rapid methods to distinguish between bioterrorism and naturally occurring plague infections are of major importance. Our study is the first to demonstrate that variable-number tandem repeats (VNTRs) in the Y. pestis genome can link human case isolates to those obtained from suspected environmental sources of infection. We demonstrate the valuable utility of VNTR markers in epidemiological investigations of naturally occurring plague and the forensic analysis of possible bioterrorism events.

The complete genome sequence of Francisella tularensis, the causative agent of tularemia.

Francisella tularensis is one of the most infectious human pathogens known. In the past, both the former Soviet Union and the US had programs to develop weapons containing the bacterium. We report the complete genome sequence of a highly virulent isolate of F. tularensis (1,892,819 bp). The sequence uncovers previously uncharacterized genes encoding type IV pili, a surface polysaccharide and iron-acquisition systems. Several virulence-associated genes were located in a putative pathogenicity island, which was duplicated in the genome. More than 10% of the putative coding sequences contained insertion-deletion or substitution mutations and seemed to be deteriorating. The genome is rich in IS elements, including IS630 Tc-1 mariner family transposons, which are not expected in a prokaryote. We used a computational method for predicting metabolic pathways and found an unexpectedly high proportion of disrupted pathways, explaining the fastidious nutritional requirements of the bacterium. The loss of biosynthetic pathways indicates that F. tularensis is an obligate host-dependent bacterium in its natural life cycle. Our results have implications for our understanding of how highly virulent human pathogens evolve and will expedite strategies to combat them.

Methods for enhanced culture recovery of Francisella tularensis.

Francisella tularensis is found in a wide variety of hosts and extrahost environments, making culture recovery a diagnostic challenge. Here we demonstrate improved recovery times and good sensitivity (90%) when cultures were inoculated on the site of an investigation using fresh tissues. For contaminated specimens, antibiotic supplementation of enriched cysteine heart agar blood culture medium improved recovery of F. tularensis by 81.1%. For transport of tissues, immediate freezing yielded culture recovery rates as high as 94%.

Laboratory analysis of tularemia in wild-trapped, commercially traded prairie dogs, Texas, 2002.

Oropharyngeal tularemia was identified as the cause of a die-off in captured wild prairie dogs at a commercial exotic animal facility in Texas. From this point source, Francisella tularensis-infected prairie dogs were traced to animals distributed to the Czech Republic and to a Texas pet shop. F. tularensis culture isolates were recovered tissue specimens from 63 prairie dogs, including one each from the secondary distribution sites. Molecular and biochemical subtyping indicated that all isolates were F. tularensis subsp. holarctica (Type B). Microagglutination assays detected antibodies against F. tularensis, with titers as great as 1:4,096 in some live animals. All seropositive animals remained culture positive, suggesting that prairie dogs may act as chronic carriers of F. tularensis. These findings demonstrate the need for additional studies of tularemia in prairie dogs, given the seriousness of the resulting disease, the fact that prairie dogs are sold commercially as pets, and the risk for pet-to-human transmission.

First reported prairie dog-to-human tularemia transmission, Texas, 2002.

A tularemia outbreak, caused by Francisella tularensis type B, occurred among wild-caught, commercially traded prairie dogs. F. tularensis microagglutination titers in one exposed person indicated recent infection. These findings represent the first evidence for prairie-dog-to-human tularemia transmission and demonstrate potential human health risks of the exotic pet trade.

An outbreak of Francisella tularensis in captive prairie dogs: an immunohistochemical analysis.

An immunohistochemical assay was developed and tested for detection of Francisella tularensis lipopolysaccaride antigen in tissues of captive prairie dogs (Cynomys ludovicianus). Tissues from 59 cases of F. tularensis were examined by this technique, which was corroborated by direct fluorescent antibody assay and direct isolation of the organism. In infected prairie dogs, studies indicated multiple, severe, necroprurulent foci occurring in the liver, lung, spleen, terminal ileum, and mandibular lymph node. Immunohistochemical analysis of the same formalin-fixed tissues indicated the presence of F. tularensis antigen in neutrophils and macrophages of these lesions and occurring extracellularly in areas of necrosis. This report demonstrates that immunohistochemical analysis is a rapid procedure that can be used to determine the pathogenesis of F. tularensis in rodent populations.

Development of a multitarget real-time TaqMan PCR assay for enhanced detection of Francisella tularensis in complex specimens.

Tularemia is the zoonotic disease caused by the gram-negative coccobacillus Francisella tularensis. Its wide distribution in the environment poses a challenge for understanding the transmission, ecology, and epidemiology of the disease. F. tularensis is also considered a potential biological weapon due to its extreme infectivity. We have developed a multitarget real-time TaqMan PCR assay capable of rapidly and accurately detecting F. tularensis in complex specimens. Targeted regions included the ISFtu2 element and the 23kDa, fopA, and tul4 genes. Analysis of the four TaqMan assays demonstrated that three (ISFtu2, 23kDa, and tul4) performed within our established criterion of a detection limit of one organism. The combined use of the three assays was highly specific, displaying no cross-reactivity with the non-Francisella bacteria tested and capable of differentially diagnosing both F. tularensis and Francisella philomiragia. When the multitarget TaqMan assay (ISFtu2, 23kDa, and tul4) was compared to culturing, using environmentally contaminated specimens, the TaqMan PCR assay was significantly more sensitive than culturing (P

Detection of novel Bartonella strains and Yersinia pestis in prairie dogs and their fleas (Siphonaptera: Ceratophyllidae and Pulicidae) using multiplex polymerase chain reaction.

We developed a multiplex polymerase chain reaction (PCR) assay that simultaneously detects three types of flea-associated microorganisms. Targets for the assay were sequences encoding portions of the gltA, a 17-kDa antigen, and pla genes of Bartonella spp. Strong et al., Rickettsia spp. da Rocha-Lima, and Yersinia pestis Yersin, respectively. A total of 260 flea samples containing bloodmeal remnants were analyzed from fleas collected from abandoned prairie dog (Cynomys ludovicianus) burrows at the site of an active plague epizootic in Jefferson County, CO. Results indicated that 34 (13.1%) fleas were positive for Bartonella spp., 0 (0%) were positive for Rickettsia spp., and 120 (46.2%) were positive for Y. pestis. Twenty-three (8.8%) of these fleas were coinfected with Bartonella spp. and Y. pestis. A second group of 295 bloodmeal-containing fleas was collected and analyzed from abandoned burrows in Logan County, CO, where a prairie dog die-off had occurred 2-4 mo before the time of sampling. Of these 295 fleas, 7 (2.4%) were positive for Bartonella spp., 0 (0%) were positive for Rickettsia spp., and 46 (15.6%) were positive for Y. pestis. Coinfections were not observed in fleas from the Logan County epizootic site. The multiplex PCR also was used to identify Y. pestis and Bartonella in prairie dog blood and tissues. This report represents the first identification of Bartonella from prairie dogs and their fleas. Prairie dog fleas were tested with PCR, and the Bartonella PCR amplicons produced were sequenced and found to be closely related to similar sequences amplified from Bartonella that had been isolated from prairie dog blood samples. Phylogenetic analyses indicate that the sequences of bartonellae from prairie dogs and prairie dog fleas cluster tightly within a clade that is distinct from those containing other known Bartonella genotypes.

Characterization of a novicida-like subspecies of Francisella tularensis isolated in Australia.

Francisella tularensis is found throughout the Northern Hemisphere, where it is associated with the disease of tularaemia in animals and humans. The isolation and identification is reported of a novicida-like subspecies of F. tularensis from a foot wound sustained in brackish water in the Northern Territory of Australia.