Targeting the Wnt signaling pathway through R-spondin 3 identifies an anti-fibrosis treatment strategy for multiple organs.

The Wnt/ß-catenin signaling pathway has been implicated in human proliferative diseases such as cancer and fibrosis. The functions of ß-catenin and several other components of this pathway have been investigated in fibrosis. However, the potential role of R-spondin proteins (RSPOs), enhancers of the Wnt/ß-catenin signaling, has not been described. A specific interventional strategy targeting this pathway for fibrosis remains to be defined. We developed monoclonal antibodies against members of the RSPO family (RSPO1, 2, and 3) and probed their potential function in fibrosis in vivo. We demonstrated that RSPO3 plays a critical role in the development of fibrosis in multiple organs. Specifically, an anti-RSPO3 antibody, OMP-131R10, when dosed therapeutically, attenuated fibrosis in carbon tetrachloride (CCl4)-induced liver fibrosis, bleomycin-induced pulmonary and skin fibrosis models. Mechanistically, we showed that RSPO3 induces multiple pro-fibrotic chemokines and cytokines in Kupffer cells and hepatocytes. We found that the anti-fibrotic activity of OMP-131R10 is associated with its inhibition of ß-catenin activation in vivo. Finally, RSPO3 was found to be highly elevated in the active lesions of fibrotic tissues in mouse models of fibrosis and in patients with idiopathic pulmonary fibrosis (IPF) and nonalcoholic steatohepatitis (NASH). Together these data provide an anti-fibrotic strategy for targeting the Wnt/ß-catenin pathway through RSPO3 blockade and support that OMP-131R10 could be an important therapeutic agent for fibrosis.

Leveraging gene expression subgroups to classify DLBCL patients and select for clinical benefit from a novel agent.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease, commonly described by cell-of-origin (COO) molecular subtypes. We sought to identify novel patient subgroups through an unsupervised analysis of a large public dataset of gene expression profiles from newly diagnosed de novo DLBCL patients, yielding 2 biologically distinct subgroups characterized by differences in the tumor microenvironment. Pathway analysis and immune deconvolution algorithms identified higher B-cell content and a strong proliferative signal in subgroup A and enriched T-cell, macrophage, and immune/inflammatory signals in subgroup B, reflecting similar biology to published DLBCL stratification research. A gene expression classifier, featuring 26 gene expression scores, was derived from the public dataset to discriminate subgroup A (classifier-negative, immune-low) and subgroup B (classifier-positive, immune-high) patients. Subsequent application to an independent series of diagnostic biopsies replicated the subgroups, with immune cell composition confirmed via immunohistochemistry. Avadomide, a CRL4CRBN E3 ubiquitin ligase modulator, demonstrated clinical activity in relapsed/refractory DLBCL patients, independent of COO subtypes. Given the immunomodulatory activity of avadomide and the need for a patient-selection strategy, we applied the gene expression classifier to pretreatment biopsies from relapsed/refractory DLBCL patients receiving avadomide (NCT01421524). Classifier-positive patients exhibited an enrichment in response rate and progression-free survival of 44% and 6.2 months vs 19% and 1.6 months for classifier-negative patients (hazard ratio, 0.49; 95% confidence interval, 0.280-0.86; P = .0096). The classifier was not prognostic for rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone or salvage immunochemotherapy. The classifier described here discriminates DLBCL tumors based on tumor and nontumor composition and has potential utility to enrich for clinical response to immunomodulatory agents, including avadomide.

SALL4 mediates teratogenicity as a thalidomide-dependent cereblon substrate.

Targeted protein degradation via small-molecule modulation of cereblon offers vast potential for the development of new therapeutics. Cereblon-binding therapeutics carry the safety risks of thalidomide, which caused an epidemic of severe birth defects characterized by forelimb shortening or phocomelia. Here we show that thalidomide is not teratogenic in transgenic mice expressing human cereblon, indicating that binding to cereblon is not sufficient to cause birth defects. Instead, we identify SALL4 as a thalidomide-dependent cereblon neosubstrate. Human mutations in SALL4 cause Duane-radial ray, IVIC, and acro-renal-ocular syndromes with overlapping clinical presentations to thalidomide embryopathy, including phocomelia. SALL4 is degraded in rabbits but not in resistant organisms such as mice because of SALL4 sequence variations. This work expands the scope of cereblon neosubstrate activity within the formerly 'undruggable' C2H2 zinc finger family and offers a path toward safer therapeutics through an improved understanding of the molecular basis of thalidomide-induced teratogenicity.

Detection of equine arteritis virus by two chromogenic RNA in situ hybridization assays (conventional and RNAscope(®)) and assessment of their performance in tissues from aborted equine fetuses.

Equine arteritis virus (EAV) is the causative agent of equine viral arteritis, a respiratory and reproductive disease of equids. EAV infection can induce abortion in pregnant mares, fulminant bronchointerstitial pneumonia in foals, and persistent infection in stallions. Here, we developed two RNA in situ hybridization (ISH) assays (conventional and RNAscope(®) ISH) for the detection of viral RNA in formalin-fixed paraffin-embedded (FFPE) tissues and evaluated and compared their performance with nucleocapsid-specific immunohistochemistry (IHC) and virus isolation (VI; gold standard) techniques. The distribution and cellular localization of EAV RNA and antigen were similar in tissues from aborted equine fetuses. Evaluation of 80 FFPE tissues collected from 16 aborted fetuses showed that the conventional RNA ISH assay had a significantly lower sensitivity than the RNAscope(®) and IHC assays, whereas there was no difference between the latter two assays. The use of oligonucleotide probes along with a signal amplification system (RNAscope(®)) can enhance detection of EAV RNA in FFPE tissues, with sensitivity comparable to that of IHC. Most importantly, these assays provide important tools with which to investigate the mechanisms of EAV pathogenesis.

Comparison of Monkeypox Virus Clade Kinetics and Pathology within the Prairie Dog Animal Model Using a Serial Sacrifice Study Design.

Monkeypox virus (MPXV) infection of the prairie dog is valuable to studying systemic orthopoxvirus disease. To further characterize differences in MPXV clade pathogenesis, groups of prairie dogs were intranasally infected (8 × 10(3) p.f.u.) with Congo Basin (CB) or West African (WA) MPXV, and 28 tissues were harvested on days 2, 4, 6, 9, 12, 17, and 24 postinfection. Samples were evaluated for the presence of virus and gross and microscopic lesions. Virus was recovered from nasal mucosa, oropharyngeal lymph nodes, and spleen earlier in CB challenged animals (day 4) than WA challenged animals (day 6). For both groups, primary viremia (indicated by viral DNA) was seen on days 6-9 through day 17. CB MPXV spread more rapidly, accumulated to greater levels, and caused greater morbidity in animals compared to WA MPXV. Histopathology and immunohistochemistry (IHC) findings, however, were similar. Two animals that succumbed to disease demonstrated abundant viral antigen in all organs tested, except for brain. Dual-IHC staining of select liver and spleen sections showed that apoptotic cells (identified by TUNEL) tended to colocalize with poxvirus antigen. Interestingly splenocytes were labelled positive for apoptosis more often than hepatocytes in both MPXV groups. These findings allow for further characterization of differences between MPXV clade pathogenesis, including identifying sites that are important during early viral replication and cellular response to viral infection.

Identification of Giardia duodenalis and Enterocytozoon bieneusi in an epizoological investigation of a laboratory colony of prairie dogs, Cynomys ludovicianus.

Since 2005, black-tailed prairie dogs (Cynomys ludovicianus) have been collected for use as research animals from field sites in Kansas, Colorado, and Texas. In January of 2012, Giardia trophozoites were identified by histology, thin-section electron microscopy, and immunofluorescent staining in the lumen of the small intestine and colon of a prairie dog euthanized because of extreme weight loss. With giardiasis suspected as the cause of weight loss, a survey of Giardia duodenalis in the laboratory colony of prairie dogs was initiated. Direct immunofluorescent testing of feces revealed active shedding of Giardia cysts in 40% (n=60) of animals held in the vivarium. All tested fecal samples (n=29) from animals in another holding facility where the index case originated were PCR positive for G. duodenalis with assemblages A and B identified from sequencing triosephosphate isomerase (tpi), glutamate dehydrogenase (gdh), and ß-giardin (bg) genes. Both assemblages are considered zoonotic, thus the parasites in prairie dogs are potential human pathogens and indicate prairie dogs as a possible wildlife reservoir or the victims of pathogen spill-over. Molecular testing for other protozoan gastrointestinal parasites revealed no Cryptosporidium infections but identified a host-adapted Enterocytozoon bieneusi genotype group.

Intraventricular granulomatous mass associated with Mycobacterium haemophilum: A rare central nervous system manifestation in a patient with human immunodeficiency virus infection.

We report a rare case of Mycobacterium haemophilum presenting as an intraventricular granulomatous mass with loculated hydrocephalus and seizures in a patient with human immunodeficiency virus. M. haemophilum, a slow-growing mycobacteria, causes localized and disseminated disease among immunocompromised hosts. Central nervous system infection with M. haemophilum is extremely rare. Preoperative laboratory testing of our patient for tuberculosis, toxoplasmosis, sarcoidosis and histoplasmosis were negative. Surgical resection of the mass revealed a caseating granuloma that stained positive for acid-fast bacillus suggesting possible tuberculoma. Despite negative testing for tuberculosis, a polymerase chain reaction analysis was ultimately performed from the resected mass which revealed M. haemophilum. To our knowledge, this is the first case of M. haemophilum presenting as an intraventricular mass. We review the clinical manifestations of this pathogen and discuss the medical and surgical management.

Novel poxvirus infection in 2 patients from the United States.

BACKGROUND: Some human poxvirus infections can be acquired through zoonotic transmission. We report a previously unknown poxvirus infection in 2 patients, 1 of whom was immunocompromised; both patients had known equine contact. METHODS: The patients were interviewed and clinical information was abstracted from the patients' medical files. Biopsies of the skin lesions were collected from both patients for histopathology, immunohistochemistry, and transmission electron microscopy analysis. Oral and skin swabs were collected from animals with frequent contact with the patients, and environmental sampling including rodent trapping was performed on the farm where the immunosuppressed patient was employed. "Pan-pox and high Guanine-cytosine" polymerase chain reaction assays were performed on patient, animal, and environmental isolates. Amplicon sequences of the viral DNA were used for agent identification and phylogenetic analysis. RESULTS: Specimens from both human cases revealed a novel poxvirus. The agent shares 88% similarity to viruses in the Parapoxvirus genus and 78% to those in the Molluscipoxvirus genus but is sufficiently divergent to resist classification as either. All animal and environmental specimens were negative for poxvirus and both patients had complete resolution of lesions. CONCLUSIONS: This report serves as a reminder that poxviruses should be considered in cutaneous human infections, especially in individuals with known barnyard exposures. The clinical course of the patients was similar to that of parapoxvirus infections, and the source of this virus is currently unknown but is presumed to be zoonotic. This report also demonstrates the importance of a comprehensive approach to diagnosis of human infections caused by previously unknown pathogens.

Notes from the field: update on Lyme carditis, groups at high risk, and frequency of associated sudden cardiac death--United States.

On December 13, 2013, MMWR published a report describing three cases of sudden cardiac death associated with Lyme carditis. State public health departments and CDC conducted a follow-up investigation to determine 1) whether carditis was disproportionately common among certain demographic groups of patients diagnosed with Lyme disease, 2) the frequency of death among patients diagnosed with Lyme disease and Lyme carditis, and 3) whether any additional deaths potentially attributable to Lyme carditis could be identified. Lyme disease cases are reported to CDC through the Nationally Notifiable Disease Surveillance System; reporting of clinical features, including Lyme carditis, is optional. For surveillance purposes, Lyme carditis is defined as acute second-degree or third-degree atrioventricular conduction block accompanying a diagnosis of Lyme disease. During 2001-2010, a total of 256,373 Lyme disease case reports were submitted to CDC, of which 174,385 (68%) included clinical information. Among these, 1,876 (1.1%) were identified as cases of Lyme carditis. Median age of patients with Lyme carditis was 43 years (range = 1-99 years); 1,209 (65%) of the patients were male, which is disproportionately larger than the male proportion among patients with other clinical manifestations (p<0.001). Of cases with this information available, 69% were diagnosed during the months of June-August, and 42% patients had an accompanying erythema migrans, a characteristic rash. Relative to patients aged 55-59 years, carditis was more common among men aged 20-39 years, women aged 25-29 years, and persons aged ≥75 years.

Acute muscular sarcocystosis: an international investigation among ill travelers returning from Tioman Island, Malaysia, 2011-2012.

BACKGROUND: Through 2 international traveler-focused surveillance networks (GeoSentinel and TropNet), we identified and investigated a large outbreak of acute muscular sarcocystosis (AMS), a rarely reported zoonosis caused by a protozoan parasite of the genus Sarcocystis, associated with travel to Tioman Island, Malaysia, during 2011-2012. METHODS: Clinicians reporting patients with suspected AMS to GeoSentinel submitted demographic, clinical, itinerary, and exposure data. We defined a probable case as travel to Tioman Island after 1 March 2011, eosinophilia (>5%), clinical or laboratory-supported myositis, and negative trichinellosis serology. Case confirmation required histologic observation of sarcocysts or isolation of Sarcocystis species DNA from muscle biopsy. RESULTS: Sixty-eight patients met the case definition (62 probable and 6 confirmed). All but 2 resided in Europe; all were tourists and traveled mostly during the summer months. The most frequent symptoms reported were myalgia (100%), fatigue (91%), fever (82%), headache (59%), and arthralgia (29%); onset clustered during 2 distinct periods: "early" during the second and "late" during the sixth week after departure from the island. Blood eosinophilia and elevated serum creatinine phosphokinase (CPK) levels were observed beginning during the fifth week after departure. Sarcocystis nesbitti DNA was recovered from 1 muscle biopsy. CONCLUSIONS: Clinicians evaluating travelers returning ill from Malaysia with myalgia, with or without fever, should consider AMS, noting the apparent biphasic aspect of the disease, the later onset of elevated CPK and eosinophilia, and the possibility for relapses. The exact source of infection among travelers to Tioman Island remains unclear but needs to be determined to prevent future illnesses.

Cerebral Angiostrongylus cantonensis infection in a captive African pygmy falcon (Polihierax semitorquatus) in southern California.

A 10-month-old, female African pygmy falcon (Polihierax semitorquatus) hatched and housed at the San Diego Zoo developed neurologic signs and died from a cerebral infection with the rat lungworm Angiostrongylus cantonensis. There was an associated mild nonsuppurative meningoencephalitis. This infection was diagnosed on histology and confirmed by detection of species-specific A. cantonensis DNA in formalin-fixed and frozen brain tissue by a polymerase chain reaction assay. To the authors' knowledge, this infection has not previously been reported in a bird in the United States and has not been known to be naturally acquired in any species in this region of the world. The source of the infection was not definitively determined but was possibly feeder geckos (Hemidactylus frenatus) imported from Southeast Asia where the parasite is endemic.

Single-dose replication-defective VSV-based Nipah virus vaccines provide protection from lethal challenge in Syrian hamsters.

Nipah virus (NiV) continues to cause outbreaks of fatal human encephalitis due to spillover from its bat reservoir. We determined that a single dose of replication-defective vesicular stomatitis virus (VSV)-based vaccine vectors expressing either the NiV fusion (F) or attachment (G) glycoproteins protected hamsters from over 1000 times LD50 NiV challenge. This highly effective single-dose protection coupled with an enhanced safety profile makes these candidates ideal for potential use in livestock and humans.

Cytokine and chemokine profiles in lung tissues from fatal cases of 2009 pandemic influenza A (H1N1): role of the host immune response in pathogenesis.

Pathological studies on fatal cases caused by 2009 pandemic influenza H1N1 virus (2009 pH1N1) reported extensive diffuse alveolar damage and virus infection predominantly in the lung parenchyma. However, the host immune response after severe 2009 pH1N1 infection is poorly understood. Herein, we investigated viral load, the immune response, and apoptosis in lung tissues from 50 fatal cases with 2009 pH1N1 virus infection. The results suggested that 7 of the 27 cytokines/chemokines showed remarkably high expression, including IL-1 receptor antagonist protein, IL-6, tumor necrosis factor-α, IL-8, monocyte chemoattractant protein-1, macrophage inflammatory protein 1-ß, and interferon-inducible protein-10 in lung tissues of 2009 pH1N1 fatal cases. Viral load, which showed the highest level on day 7 of illness onset and persisted until day 17 of illness, was positively correlated with mRNA levels of IL-1 receptor antagonist protein, monocyte chemoattractant protein-1, macrophage inflammatory protein 1-ß, interferon-inducible protein-10, and regulated on activation normal T-cell expressed and secreted. Apoptosis was evident in lung tissues stained by the TUNEL assay. Decreased Fas and elevated FasL mRNA levels were present in lung tissues, and cleaved caspase-3 was frequently seen in pneumocytes, submucosal glands, and lymphoid tissues. The pathogenesis of the 2009 pH1N1 virus infection is associated with viral replication and production of proinflammatory mediators. FasL and caspase-3 are involved in the pathway of 2009 pH1N1 virus-induced apoptosis in lung tissues, and the disequilibrium between the Fas and FasL level in lung tissues could contribute to delayed clearance of the virus and subsequent pathological damages.

Fatal transplant-associated west nile virus encephalitis and public health investigation-california, 2010.

BACKGROUND: In December 2010, a case of West Nile virus (WNV) encephalitis occurring in a kidney recipient shortly after organ transplantation was identified. METHODS: A public health investigation was initiated to determine the likely route of transmission, detect potential WNV infections among recipients from the same organ donor, and remove any potentially infected blood products or tissues. Available serum, cerebrospinal fluid, and urine samples from the organ donor and recipients were tested for WNV infection by nucleic acid testing and serology. RESULTS: Two additional recipients from the same organ donor were identified, their clinical and exposure histories were reviewed, and samples were obtained. WNV RNA was retrospectively detected in the organ donor's serum. After transplantation, the left kidney recipient had serologic and molecular evidence of WNV infection and the right kidney recipient had prolonged but clinically inapparent WNV viremia. The liver recipient showed no clinical signs of infection but had flavivirus IgG antibodies; however, insufficient samples were available to determine the timing of infection. No remaining infectious products or tissues were identified. CONCLUSIONS: Clinicians should suspect WNV as a cause of encephalitis in organ transplant recipients and report cases to public health departments for prompt investigation of the source of infection. Increased use of molecular testing and retaining pretransplantation sera may improve the ability to detect and diagnose transplant-associated WNV infection in organ transplant recipients.

Exserohilum infections associated with contaminated steroid injections: a clinicopathologic review of 40 cases.

September 2012 marked the beginning of the largest reported outbreak of infections associated with epidural and intra-articular injections. Contamination of methylprednisolone acetate with the black mold, Exserohilum rostratum, was the primary cause of the outbreak, with >13,000 persons exposed to the potentially contaminated drug, 741 confirmed drug-related infections, and 55 deaths. Fatal meningitis and localized epidural, paraspinal, and peripheral joint infections occurred. Tissues from 40 laboratory-confirmed cases representing these various clinical entities were evaluated by histopathological analysis, special stains, and IHC to characterize the pathological features and investigate the pathogenesis of infection, and to evaluate methods for detection of Exserohilum in formalin-fixed, paraffin-embedded (FFPE) tissues. Fatal cases had necrosuppurative to granulomatous meningitis and vasculitis, with thrombi and abundant angioinvasive fungi, with extensive involvement of the basilar arterial circulation of the brain. IHC was a highly sensitive method for detection of fungus in FFPE tissues, demonstrating both hyphal forms and granular fungal antigens, and PCR identified Exserohilum in FFPE and fresh tissues. Our findings suggest a pathogenesis for meningitis involving fungal penetration into the cerebrospinal fluid at the injection site, with transport through cerebrospinal fluid to the basal cisterns and subsequent invasion of the basilar arteries. Further studies are needed to characterize Exserohilum and investigate the potential effects of underlying host factors and steroid administration on the pathogenesis of infection.

Fatal wild-type varicella-zoster virus encephalitis without a rash in a vaccinated child.

Encephalitis associated with varicella-zoster virus, rare among children in the varicella vaccine era, has generally been associated with a rash. We report fatal wild-type varicella-zoster virus encephalitis without a rash in a child who had received 1 dose of varicella vaccine. Varicella-zoster virus encephalitis should be considered in the differential diagnosis for children presenting with acute neurologic symptoms, even vaccine recipients.

Localization of pandemic 2009 H1N1 influenza A virus RNA in lung and lymph nodes of fatal influenza cases by in situ hybridization: new insights on virus replication and pathogenesis.

BACKGROUND: Pandemic 2009 H1N1 influenza A (pH1N1) virus has caused substantial morbidity and mortality globally and continues to circulate. Although pH1N1 viral antigens have been demonstrated in various human tissues by immunohistochemistry (IHC), cellular localization of pH1N1 RNA in these tissues has largely remained uninvestigated. OBJECTIVES: To examine the distribution of pH1N1 RNA in tissues of fatal cases in order to understand the virus tissue tropism, replication and disease pathogenesis. STUDY DESIGN: Formalin-fixed, paraffin embedded autopsy tissues from 21 patients with confirmed pH1N1 infection were analyzed by influenza A IHC and by in situ hybridization (ISH) using DIG-labeled sense (detects viral RNA) and antisense probes (detects positive-stranded mRNA and cRNA) targeting the nucleoprotein gene of pH1N1 virus. RESULTS: pH1N1 RNA was localized by ISH in 57% of cases while viral antigens were detected by IHC in 76%. However, in cases with a short duration of illness (1-3 days), more cases (69%) were positive by ISH than IHC (62%). Strong ISH staining was detected by antisense probes in the alveolar pneumocytes of the lungs, mucous glands and in lymph nodes. IHC staining of viral antigens was demonstrated in the lung pneumocytes and mucous glands, but no immunostaining was detected in any of the lymph nodes examined. CONCLUSIONS: This study demonstrates cellular localization of positive-stranded pH1N1 RNA in the lungs, mucous glands and lymph nodes that suggests viral replication in these tissues. The novel ISH assay can be a useful adjunct for the detection of pH1N1 virus in tissues and for pathogenesis studies.

Necrotizing cutaneous mucormycosis after a tornado in Joplin, Missouri, in 2011.

BACKGROUND: Mucormycosis is a fungal infection caused by environmentally acquired molds. We investigated a cluster of cases of cutaneous mucormycosis among persons injured during the May 22, 2011, tornado in Joplin, Missouri. METHODS: We defined a case as a soft-tissue infection in a person injured during the tornado, with evidence of a mucormycete on culture or immunohistochemical testing plus DNA sequencing. We conducted a case-control study by reviewing medical records and conducting interviews with case patients and hospitalized controls. DNA sequencing and whole-genome sequencing were performed on clinical specimens to identify species and assess strain-level differences, respectively. RESULTS: A total of 13 case patients were identified, 5 of whom (38%) died. The patients had a median of 5 wounds (range, 1 to 7); 11 patients (85%) had at least one fracture, 9 (69%) had blunt trauma, and 5 (38%) had penetrating trauma. All case patients had been located in the zone that sustained the most severe damage during the tornado. On multivariate analysis, infection was associated with penetrating trauma (adjusted odds ratio for case patients vs. controls, 8.8; 95% confidence interval [CI], 1.1 to 69.2) and an increased number of wounds (adjusted odds ratio, 2.0 for each additional wound; 95% CI, 1.2 to 3.2). Sequencing of the D1-D2 region of the 28S ribosomal DNA yielded Apophysomyces trapeziformis in all 13 case patients. Whole-genome sequencing showed that the apophysomyces isolates were four separate strains. CONCLUSIONS: We report a cluster of cases of cutaneous mucormycosis among Joplin tornado survivors that were associated with substantial morbidity and mortality. Increased awareness of fungi as a cause of necrotizing soft-tissue infections after a natural disaster is warranted.

The pox in the North American backyard: Volepox virus pathogenesis in California mice (Peromyscus californicus).

Volepox virus (VPXV) was first isolated in 1985 from a hind foot scab of an otherwise healthy California vole (Microtus californicus). Subsequent surveys in San Mateo County, CA, revealed serological evidence suggesting that VPXV is endemic to this area, and a second viral isolate from a Pinyon mouse (Peromyscus truei) was collected in 1988. Since then, few studies have been conducted regarding the ecology, pathology, and pathogenicity of VPXV, and its prevalence and role as a potential zoonotic agent remain unknown. To increase our understanding of VPXV disease progression, we challenged 24 California mice (Peromyscus californicus) intranasally with 1.6 × 10(3) PFU of purified VPXV. By day five post infection (pi) we observed decreased activity level, conjunctivitis, ruffled hair, skin lesions, facial edema, and crusty noses. A mortality rate of 54% was noted by day eight pi. In addition, internal organ necrosis and hemorrhages were observed during necropsy of deceased or euthanized animals. Viral loads in tissues (brain, gonad, kidney, liver, lung, spleen, submandibular lymph node, and adrenal gland), bodily secretions (saliva, and tears), and excretions (urine, and/or feces) were evaluated and compared using real time-PCR and tissue culture. Viral loads measured as high as 2 × 10(9) PFU/mL in some organs. Our results suggest that VPXV can cause extreme morbidity and mortality within rodent populations sympatric with the known VPXV reservoirs.