Juan G. Lopera

Characterization of West Nile Viruses Isolated from Captive American Flamingoes (Phoenicopterus ruber) in Medellin, Colombia

Serum samples from a total of 71 healthy captive birds belonging to 18 species were collected in July of 2008 in Medellin (Colombia) and tested for flaviviruses. Eighteen of 29 samples from American Flamingoes (Phoenicopterus ruber) were positive for West Nile virus (WNV) by reverse transcription-polymerase chain reaction. Selected positive samples were serially passaged and WNV was confirmed by immunofluorescence. Two isolates (524/08, 9835/08) were characterized in vitro and in vivo. Sequence analysis revealed WNV with 16 nucleotide substitutions resulting in six amino acid changes when compared with the NY99 strain. Colombian (COL) viruses were more closely related to Louisiana isolates from 2001. When compared with attenuated strains isolated from Texas, COL isolates differed in their plaque size and temperature sensitivity phenotype. The COL viruses were pathogenic in embryonated chicken eggs and Balb/c mice.

Evaluation of monkeypox virus infection of black-tailed prairie dogs (Cynomys ludovicianus) using in vivo bioluminescent imaging.

Abstract Monkeypox (MPX) is a re-emerging zoonotic disease that is endemic in Central and West Africa, where it can cause a smallpox-like disease in humans. Despite many epidemiologic and field investigations of MPX, no definitive reservoir species has been identified. Using recombinant viruses expressing the firefly luciferase (luc) gene, we previously demonstrated the suitability of in vivo bioluminescent imaging (BLI) to study the pathogenesis of MPX in animal models. Here, we evaluated BLI as a novel approach for tracking MPX virus infection in black-tailed prairie dogs (Cynomys ludovicianus). Prairie dogs were affected during a multistate outbreak of MPX in the US in 2003 and have since been used as an animal model of this disease. Our BLI results were compared with PCR and virus isolation from tissues collected postmortem. Virus was easily detected and quantified in skin and superficial tissues by BLI before and during clinical phases, as well as in subclinical secondary cases, but was not reliably detected in deep tissues such as the lung. Although there are limitations to viral detection in larger wild rodent species, BLI can enhance the use of prairie dogs as an animal model of MPX and can be used for the study of infection, disease progression, and transmission in potential wild rodent reservoirs.

Further Assessment of Monkeypox Virus Infection in Gambian Pouched Rats (Cricetomys gambianus) Using In Vivo Bioluminescent Imaging.

Monkeypox is a zoonosis clinically similar to smallpox in humans. Recent evidence has shown a potential risk of increased incidence in central Africa. Despite attempts to isolate the virus from wild rodents and other small mammals, no reservoir host has been identified. In 2003, Monkeypox virus (MPXV) was accidentally introduced into the U.S. via the pet trade and was associated with the Gambian pouched rat (Cricetomys gambianus). Therefore, we investigated the potential reservoir competence of the Gambian pouched rat for MPXV by utilizing a combination of in vivo and in vitro methods. We inoculated three animals by the intradermal route and three animals by the intranasal route, with one mock-infected control for each route. Bioluminescent imaging (BLI) was used to track replicating virus in infected animals and virological assays (e.g. real time PCR, cell culture) were used to determine viral load in blood, urine, ocular, nasal, oral, and rectal swabs. Intradermal inoculation resulted in clinical signs of monkeypox infection in two of three animals. One severely ill animal was euthanized and the other affected animal recovered. In contrast, intranasal inoculation resulted in subclinical infection in all three animals. All animals, regardless of apparent or inapparent infection, shed virus in oral and nasal secretions. Additionally, BLI identified viral replication in the skin without grossly visible lesions. These results suggest that Gambian pouched rats may play an important role in transmission of the virus to humans, as they are hunted for consumption and it is possible for MPXV-infected pouched rats to shed infectious virus without displaying overt clinical signs.

Increased Biosynthetic Gene Dosage in a Genome-Reduced Defensive Bacterial Symbiont

Secondary metabolites, which are small-molecule organic compounds produced by living organisms, provide or inspire drugs for many different diseases. These natural products have evolved over millions of years to provide a survival benefit to the producing organism and often display potent biological activity with important therapeutic applications. For instance, defensive compounds in the environment may be cytotoxic to eukaryotic cells, a property exploitable for cancer treatment. Here, we describe the genome of an uncultured symbiotic bacterium that makes such a cytotoxic metabolite. This symbiont is losing genes that do not endow a selective advantage in a hospitable host environment. Secondary metabolism genes, however, are repeated multiple times in the genome, directly demonstrating their selective advantage. This finding shows the strength of selective forces in symbiotic relationships and suggests that uncultured bacteria in such relationships should be targeted for drug discovery efforts. ABSTRACT A symbiotic lifestyle frequently results in genome reduction in bacteria; the isolation of small populations promotes genetic drift and the fixation of deletions and deleterious mutations over time. Transitions in lifestyle, including host restriction or adaptation to an intracellular habitat, are thought to precipitate a wave of sequence degradation events and consequent proliferation of pseudogenes. We describe here a verrucomicrobial symbiont of the tunicate Lissoclinum sp. that appears to be undergoing such a transition, with low coding density and many identifiable pseudogenes. However, despite the overall drive toward genome reduction, this symbiont maintains seven copies of a large polyketide synthase (PKS) pathway for the mandelalides (mnd), cytotoxic compounds that likely constitute a chemical defense for the host. There is evidence of ongoing degradation in a small number of these repeats—including variable borders, internal deletions, and single nucleotide polymorphisms (SNPs). However, the gene dosage of most of the pathway is increased at least 5-fold. Correspondingly, this single pathway accounts for 19% of the genome by length and 25.8% of the coding capacity. This increased gene dosage in the face of generalized sequence degradation and genome reduction suggests that mnd genes are under strong purifying selection and are important to the symbiotic relationship. IMPORTANCE Secondary metabolites, which are small-molecule organic compounds produced by living organisms, provide or inspire drugs for many different diseases. These natural products have evolved over millions of years to provide a survival benefit to the producing organism and often display potent biological activity with important therapeutic applications. For instance, defensive compounds in the environment may be cytotoxic to eukaryotic cells, a property exploitable for cancer treatment. Here, we describe the genome of an uncultured symbiotic bacterium that makes such a cytotoxic metabolite. This symbiont is losing genes that do not endow a selective advantage in a hospitable host environment. Secondary metabolism genes, however, are repeated multiple times in the genome, directly demonstrating their selective advantage. This finding shows the strength of selective forces in symbiotic relationships and suggests that uncultured bacteria in such relationships should be targeted for drug discovery efforts. Author Video: An author video summary of this article is available.

Characterization of Monkeypox virus infection in African rope squirrels (Funisciurus sp.)

Monkeypox (MPX) is a zoonotic disease endemic in Central and West Africa and is caused by Monkeypox virus (MPXV), the most virulent Orthopoxvirus affecting humans since the eradication of Variola virus (VARV). Many aspects of the MPXV transmission cycle, including the natural host of the virus, remain unknown. African rope squirrels (Funisciurus spp.) are considered potential reservoirs of MPXV, as serosurveillance data in Central Africa has confirmed the circulation of the virus in these rodent species [1,2]. In order to understand the tissue tropism and clinical signs associated with infection with MPXV in these species, wild-caught rope squirrels were experimentally infected via intranasal and intradermal exposure with a recombinant MPXV strain from Central Africa engineered to express the luciferase gene. After infection, we monitored viral replication and shedding via in vivo bioluminescent imaging, viral culture and real time PCR. MPXV infection in African rope squirrels caused mortality and moderate to severe morbidity, with clinical signs including pox lesions in the skin, eyes, mouth and nose, dyspnea, and profuse nasal discharge. Both intranasal and intradermal exposures induced high levels of viremia, fast systemic spread, and long periods of viral shedding. Shedding and luminescence peaked at day 6 post infection and was still detectable after 15 days. Interestingly, one sentinel animal, housed in the same room but in a separate cage, also developed severe MPX disease and was euthanized. This study indicates that MPXV causes significant pathology in African rope squirrels and infected rope squirrels shed large quantities of virus, supporting their role as a potential source of MPXV transmission to humans and other animals in endemic MPX regions.

Attenuation of monkeypox virus by deletion of genomic regions

Monkeypox virus (MPXV) is an emerging pathogen from Africa that causes disease similar to smallpox. Two clades with different geographic distributions and virulence have been described. Here, we utilized bioinformatic tools to identify genomic regions in MPXV containing multiple virulence genes and explored their roles in pathogenicity; two selected regions were then deleted singularly or in combination. In vitro and in vivo studies indicated that these regions play a significant role in MPXV replication, tissue spread, and mortality in mice. Interestingly, while deletion of either region led to decreased virulence in mice, one region had no effect on in vitro replication. Deletion of both regions simultaneously also reduced cell culture replication and significantly increased the attenuation in vivo over either single deletion. Attenuated MPXV with genomic deletions present a safe and efficacious tool in the study of MPX pathogenesis and in the identification of genetic factors associated with virulence.

Autometa: Automated extraction of microbial genomes from individual shotgun metagenomes

Motivation Shotgun metagenomics is a powerful, high-resolution technique enabling the study of microbial communities in situ. However, species-level resolution is only achieved after a process of “binning” where contigs predicted to originate from the same genome are clustered. Such culture-independent sequencing frequently unearths novel microbes, and so various methods have been devised for reference-free binning. Existing methods, however, suffer from: (1) reliance on human pattern recognition, which is inherently unscalable; (2) requirement for multiple co-assembled metagenomes, which degrades assembly quality due to strain variance; and (3) assumption of prior host genome removal not feasible for non-model hosts. We therefore devised a fully-automated pipeline, termed “Autometa,” to address these issues. Results: Autometa implements a method for taxonomic partitioning of contigs based on predicted protein homology, and this was shown to vastly improve binning in host-associated and complex metagenomes. Autometa’s method of automated clustering, based on Barnes-Hut Stochastic Neighbor Embedding (BH-tSNE) and DBSCAN, was shown to be highly scalable, outperforming other binning pipelines in complex simulated datasets. Availability and implementation Autometa is freely available at https://bitbucket.org/jasonckwan/autometa and as a docker image at https://hub.docker.com/r/jasonkwan/autometa under the GNU Affero General Public License 3 (AGPL 3). Contact jason.kwan@wisc.edu Supplementary information Supplementary data are available attached to this article at https://biorxiv.org

Monkeypox challenge of rope squirrels: data

Monkeypox virus (MPXV) is a zoonotic disease endemic in Central and West Africa and is the most virulent orthopoxvirus affecting humans since the eradication of variola virus (VARV). In order to assess their reservoir potential, wild-caught rope squirrels were experimentally infected via intranasal and intradermal exposure with a recombinant MPXV strain from Central Africa engineered to express the luciferase gene. After infection, we monitored viral replication via in vivo bioluminescent imaging and viral shedding via culture and PCR. MPXV infection in African rope squirrels caused mortality and moderate to severe morbidity, with clinical signs including pox lesions in the skin, eyes, mouth and nose, dyspnea, and profuse nasal discharge. Both intranasal and intradermal exposures induced high levels of viremia, fast systemic spread, and long periods of viral shedding. Shedding and luminescence peaked at day 6 post infection and was still detectable after 15 days. Evidence of viral persistence in tissues by real-time PCR was observed in animals that survived infection. Interestingly, one sentinel animal, housed in the same room but in a separate cage, also developed severe MPX disease and was euthanized. This study highlights the epidemiological importance of African rope squirrels and suggests the potential role of this rodent species as a natural reservoir host of MPXV and a source of direct transmission to humans and other animals in endemic MPX regions.