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Human alphaherpesvirus 1 (HuAHV1) causes fatal neurologic infections in captive New World primates. To determine risks for interspecies transmission, we examined data for 13 free-ranging, black-tufted marmosets (Callithrix penicillata) that died of HuAHV1 infection and had been in close contact with humans in anthropized areas in Brazil during 2012–2019. We evaluated pathologic changes in the marmosets, localized virus and antigen, and assessed epidemiologic features. The main clinical findings were neurologic signs, necrotizing meningoencephalitis, and ulcerative glossitis; 1 animal had necrotizing hepatitis. Transmission electron microscopy revealed intranuclear herpetic inclusions, and immunostaining revealed HuAHV1 and herpesvirus particles in neurons, glial cells, tongue mucosal epithelium, and hepatocytes. PCR confirmed HuAHV1 infection. These findings illustrate how disruption of the One Health equilibrium in anthropized environments poses risks for interspecies virus transmission with potential spillover not only from animals to humans but also from humans to free-ranging nonhuman primates or other animals.

Severe coronavirus disease in neonates is rare. We analyzed clinical, laboratory, and autopsy findings from a neonate in the United States who was delivered at 25 weeks of gestation and died 4 days after birth; the mother had asymptomatic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and preeclampsia. We observed severe diffuse alveolar damage and localized SARS-CoV-2 by immunohistochemistry, in situ hybridization, and electron microscopy of the lungs of the neonate. We localized SARS-CoV-2 RNA in neonatal heart and liver vascular endothelium by using in situ hybridization and detected SARS-CoV-2 RNA in neonatal and placental tissues by using reverse transcription PCR. Subgenomic reverse transcription PCR suggested viral replication in lung/airway, heart, and liver. These findings indicate that in utero SARS-CoV-2 transmission contributed to this neonatal death.

Efforts to combat the coronavirus disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have placed a renewed focus on the use of transmission electron microscopy for identifying coronavirus in tissues. In attempts to attribute pathology of COVID-19 patients directly to tissue damage caused by SARS-CoV-2, investigators have inaccurately reported subcellular structures, including coated vesicles, multivesicular bodies, and vesiculating rough endoplasmic reticulum, as coronavirus particles. We describe morphologic features of coronavirus that distinguish it from subcellular structures, including particle size range (60–140 nm), intracellular particle location within membrane-bound vacuoles, and a nucleocapsid appearing in cross section as dense dots (6–12 nm) within the particles. In addition, although the characteristic spikes of coronaviruses may be visible on the virus surface, especially on extracellular particles, they are less evident in thin sections than in negative stain preparations.

Arenaviruses, such as Lassa virus (LASV), can cause severe and fatal hemorrhagic fevers (e.g., Lassa fever, LF) in humans with no vaccines or therapeutics. Research on arenavirus-induced hemorrhagic fevers (AHFs) has been hampered by the highly virulent nature of these viral pathogens, which require high biocontainment laboratory, and the lack of an immune-competent small animal model that can recapitulate AHF disease and pathological features. Guinea pig infected with Pichinde virus (PICV), an arenavirus that does not cause disease in humans, has been established as a convenient surrogate animal model for AHFs as it can be handled in a conventional laboratory. The PICV strain P18, derived from sequential passaging of the virus 18 times in strain 13 inbred guinea pigs, causes severe febrile illness in guinea pigs that is reminiscent of lethal LF in humans. As inbred guinea pigs are not readily available and are difficult to maintain, outbred Hartley guinea pigs have been used but they show a high degree of disease heterogeneity upon virulent P18 PICV infection. Here, we describe an improved outbred guinea-pig infection model using recombinant rP18 PICV generated by reverse genetics technique followed by plaque purification, which consistently shows >90% mortality and virulent infection. Comprehensive virological, histopathological, and immunohistochemical analyses of the rP18-virus infected animals show similar features of human LASV infection. Our data demonstrate that this improved animal model can serve as a safe, affordable, and convenient surrogate small animal model for studying human LF pathogenesis and for evaluating efficacy of preventative or therapeutic approaches.

An ongoing pandemic of coronavirus disease (COVID-19) is caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Characterization of the histopathology and cellular localization of SARS-CoV-2 in the tissues of patients with fatal COVID-19 is critical to further understand its pathogenesis and transmission and for public health prevention measures. We report clinicopathologic, immunohistochemical, and electron microscopic findings in tissues from 8 fatal laboratory-confirmed cases of SARS-CoV-2 infection in the United States. All cases except 1 were in residents of long-term care facilities. In these patients, SARS-CoV-2 infected epithelium of the upper and lower airways with diffuse alveolar damage as the predominant pulmonary pathology. SARS-CoV-2 was detectable by immunohistochemistry and electron microscopy in conducting airways, pneumocytes, alveolar macrophages, and a hilar lymph node but was not identified in other extrapulmonary tissues. Respiratory viral co-infections were identified in 3 cases; 3 cases had evidence of bacterial co-infection.

Zika virus (ZIKV) can infect the central nervous system, but it is not clear how it reaches the brain. Here, Ayala-Nunez et al. show in ex vivo and in vivo models that ZIKV can hitch a ride in monocytes in a Trojan Horse manner to cross the endothelium and disseminate the virus.

In late September 2017, Bwabwata National Park in Namibia experienced a sudden die-off of hippopotamuses and Cape buffalo. A multiorganizational response was initiated, involving several ministries within Namibia and the US Centers for Disease Control and Prevention. Rapid interventions resulted in zero human or livestock cases associated with this epizootic.

Lassa virus (LASV), a hemorrhagic fever virus endemic to West Africa, causes conjunctivitis in patients with acute disease. To examine ocular manifestations of LASV, we histologically examined eyes from infected guinea pigs. In fatal disease, LASV immunostaining was most prominent in the anterior uvea, especially in the filtration angle, ciliary body, and iris and in and around vessels in the bulbar conjunctiva and peripheral cornea, where it co-localized with an endothelial marker (platelet endothelial cell adhesion molecule). Antigen was primarily associated with infiltration of T-lymphocytes around vessels in the anterior uvea and with new vessel formation at the peripheral cornea. In animals that exhibited clinical signs but survived infection, eyes had little to no inflammation and no LASV immunostaining 6 weeks after infection. Overall, in this model, LASV antigen was restricted to the anterior uvea and was associated with mild chronic inflammation in animals with severe disease but was not detected in survivors.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) shares common clinicopathologic features with other severe pulmonary illnesses. Hantavirus pulmonary syndrome was diagnosed in 2 patients in Arizona, USA, suspected of dying from infection with SARS-CoV-2. Differential diagnoses and possible co-infections should be considered for cases of respiratory distress during the SARS-CoV-2 pandemic.

Ebola virus disease (EVD) is associated with elevated cytokine levels, and hypercytokinemia is more pronounced in fatal cases. This type of hyperinflammatory state is reminiscent of 2 rheumatologic disorders known as macrophage activation syndrome and hemophagocytic lymphohistiocytosis, which are characterized by macrophage and T-cell activation. An evaluation of 2 cohorts of patients with EVD revealed that a marker of macrophage activation (sCD163) but not T-cell activation (sCD25) was associated with severe and fatal EVD. Furthermore, substantial immunoreactivity of host tissues to a CD163-specific antibody, predominantly in areas of extensive immunostaining for Ebola virus antigens, was observed in fatal cases. These data suggest that host macrophage activation contributes to EVD pathogenesis and that directed antiinflammatory therapies could be beneficial in the treatment of EVD.

Crimean-Congo hemorrhagic fever virus (CCHFV, order Bunyavirales, family Nairoviridae, genus Orthonairovirus) is the tick-borne etiological agent of Crimean-Congo hemorrhagic fever (CCHF) in humans. Animals are generally susceptible to CCHFV infection but refractory to disease. Small animal models are limited to interferon-deficient mice, that develop acute fatal disease following infection. Here, using a ZsGreen1- (ZsG) expressing reporter virus (CCHFV/ZsG), we examine tissue tropism and dissemination of virus in interferon-α/β receptor knock-out (Ifnar-/-) mice. We demonstrate that CCHFV/ZsG retains in vivo pathogenicity comparable to wild-type virus. Interestingly, despite high levels of viral RNA in all organs assessed, 2 distribution patterns of infection were observed by both fluorescence and immunohistochemistry (IHC), corresponding to the permissiveness of organ tissues. To further investigate viral dissemination and to temporally define cellular targets of CCHFV in vivo, mice were serially euthanized at different stages of disease. Flow cytometry was used to characterize CCHFV-associated alterations in hematopoietic cell populations and to classify infected cells in the blood, lymph node, spleen, and liver. ZsG signal indicated that mononuclear phagocytic cells in the lymphatic tissues were early targets of infection; in late-stage infection, overall, the highest levels of signal were detected in the liver, and ZsG was found in both antigen-presenting and lymphocyte cell populations.

Circulation of Zika virus (ZIKV) was first identified in the Western hemisphere in late 2014. Primarily transmitted through mosquito bite, ZIKV can also be transmitted through sex and from mother to fetus, and maternal ZIKV infection has been associated with fetal malformations. We assessed immunodeficient AG129 mice for their capacity to shed ZIKV in semen and to infect female mice via sexual transmission. Infectious virus was detected in semen between 7 and 21 days post-inoculation, and ZIKV RNA was detected in semen through 58 days post-inoculation. During mating, 73% of infected males transmitted ZIKV to uninfected females, and 50% of females became infected, with evidence of fetal infection in resulting pregnancies. Semen from vasectomized mice contained significantly lower levels of infectious virus, though sexual transmission still occurred. This model provides a platform for studying the kinetics of ZIKV sexual transmission and prolonged RNA shedding also observed in human semen.

Lassa virus (LASV) is a mammarenavirus (arenavirus) that causes zoonotic infection in humans that can lead to fatal hemorrhagic Lassa fever (LF) disease. Currently, there are no FDA-approved vaccines or therapeutics against LASV. Development of treatments against LF and other related arenavirus-induced hemorrhagic fevers (AHFs) requires relevant animal models that can recapitulate clinical and pathological features of AHF diseases in humans. Laboratory mice are generally resistant to LASV infection, and non-human primates, while being a good animal model for LF, are limited by their high cost. Here, we describe a small, affordable, and convenient animal model that is based on outbred Hartley guinea pigs infected with Pichinde virus (PICV), a mammarenavirus that is non-pathogenic in humans, for use as a surrogate model of human LF. We conducted a detailed analysis of tissue histopathology and immunohistochemical analysis of different organs of outbred Hartley guinea pigs infected with different PICV strains that show differential disease phenotypes and pathologies. Comparing to infection with the avirulent PICV strain (P2 or rP2), animals infected with the virulent strain (P18 or rP18) show extensive pathological changes in different organs that sustain high levels of virus replication. The similarity of tissue pathology and viral antigen distribution between the virulent PICV–guinea pig model and lethal human LASV infection supports a role of this small animal model as a surrogate model of studying human LF in order to understand its pathogenesis and for evaluating potential preventative and therapeutic options against AHFs.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tri-segmented, tick-borne nairovirus that causes disease of ranging severity in humans. The CCHFV M segment encodes a complex glycoprotein precursor (GPC) that undergoes extensive endoproteolytic cleavage, giving rise to two structural proteins (Gn and Gc) required for virus attachment and entry, and to multiple non-structural proteins (NSm, GP160, GP85, and GP38). The functions of these non-structural proteins remain largely unclear. Here, we investigate the role of NSm during infection by generating a recombinant CCHFV lacking the complete NSm domain (10200∆NSm) and observing CCHFV ∆NSm replication in cell lines and pathogenicity in Ifnar-/- mice. Our data demonstrate that the NSm domain is dispensable for viral replication in vitro, and, despite the delayed onset of clinical signs, CCHFV lacking this domain caused severe or lethal disease in infected mice.

Background: An estimated 100,000 cases of congenital rubella syndrome (CRS) occur worldwide each year. The reported mortality rate for infants with CRS is up to 33%. The cellular mechanisms responsible for the multiple congenital defects in CRS are presently unknown. Here we identify cell types positive for rubella virus (RV) in CRS infants. Methods: Cells and organs involved in RV replication were identified in paraffin-embedded autopsy tissues from three fatal case-patients by histopathologic examination and immunohistochemical (IHC) staining using a rabbit polyclonal RV antibody. Normal rabbit antisera and RV antisera preabsorbed with highly purified RV served as negative controls. Results: RV antigen was found in interstitial fibroblasts in the heart, adventitial fibroblasts of large blood vessels, alveolar macrophages, progenitor cells of the outer granular layer of the brain, and in capillary endothelium and basal plate in the placenta. The antibody specificity was verified by IHC staining of multiple tissue sections from other infectious disease cases. RV infection of each cell type is consistent with abnormalities which have been identified in patients with CRS, in the heart, large blood vessels, and brain. Antigen distribution was consistent with inflammatory response to vascular injury and systemic spread of RV. Conclusions: The identification of RV positive cell types in CRS is important to better understand the pathology and pathogenesis of CRS.

The Egyptian rousette bat (Rousettus aegyptiacus) is a natural reservoir for marburgviruses and a consistent source of virus spillover to humans. Cumulative evidence suggests various bat species may also transmit ebolaviruses. We investigated the susceptibility of Egyptian rousettes to each of the five known ebolaviruses (Sudan, Ebola, Bundibugyo, Taï Forest, and Reston), and compared findings with Marburg virus. In a pilot study, groups of four juvenile bats were inoculated with one of the ebolaviruses or Marburg virus. In ebolavirus groups, viral RNA tissue distribution was limited, and no bat became viremic. Sudan viral RNA was slightly more widespread, spurring a second, 15-day Sudan virus serial euthanasia study. Low levels of Sudan viral RNA disseminated to multiple tissues at early time points, but there was no viremia or shedding. In contrast, Marburg virus RNA was widely disseminated, with viremia, oral and rectal shedding, and antigen in spleen and liver. This is the first experimental infection study comparing tissue tropism, viral shedding, and clinical and pathologic effects of six different filoviruses in the Egyptian rousette, a known marburgvirus reservoir. Our results suggest Egyptian rousettes are unlikely sources for ebolaviruses in nature, and support a possible single filovirus—single reservoir host relationship.

Egyptian rousette bats (Rousettus aegyptiacus) are natural reservoir hosts of Marburg virus (MARV), and Ravn virus (RAVV; collectively called marburgviruses) and have been linked to human cases of Marburg virus disease (MVD). We investigated the clinical and pathologic effects of experimental MARV infection in Egyptian rousettes through a serial euthanasia study and found clear evidence of mild but transient disease. Three groups of nine, captive-born, juvenile male bats were inoculated subcutaneously with 10,000 TCID50 of Marburg virus strain Uganda 371Bat2007, a minimally passaged virus originally isolated from a wild Egyptian rousette. Control bats (n = 3) were mock-inoculated. Three animals per day were euthanized at 3, 5–10, 12 and 28 days post-inoculation (DPI); controls were euthanized at 28 DPI. Blood chemistry analyses showed a mild, statistically significant elevation in alanine aminotransferase (ALT) at 3, 6 and 7 DPI. Lymphocyte and monocyte counts were mildly elevated in inoculated bats after 9 DPI. Liver histology revealed small foci of inflammatory infiltrate in infected bats, similar to lesions previously described in wild, naturally-infected bats. Liver lesion severity scores peaked at 7 DPI, and were correlated with both ALT and hepatic viral RNA levels. Immunohistochemical staining detected infrequent viral antigen in liver (3–8 DPI, n = 8), spleen (3–7 DPI, n = 8), skin (inoculation site; 3–12 DPI, n = 20), lymph nodes (3–10 DPI, n = 6), and oral submucosa (8–9 DPI, n = 2). Viral antigen was present in histiocytes, hepatocytes and mesenchymal cells, and in the liver, antigen staining co-localized with inflammatory foci. These results show the first clear evidence of very mild disease caused by a filovirus in a reservoir bat host and provide support for our experimental model of this virus-reservoir host system.

During outbreaks of infectious diseases or in cases of severely ill patients, it is imperative to identify the causative agent. This report describes several events in which virus isolation and identification by electron microscopy were critical to initial recognition of the etiologic agent, which was further analyzed by additional laboratory diagnostic assays. Examples include severe acute respiratory syndrome coronavirus, and Nipah, lymphocytic choriomeningitis, West Nile, Cache Valley, and Heartland viruses. These cases illustrate the importance of the techniques of cell culture and electron microscopy in pathogen identification and recognition of emerging diseases.

Dengue is a leading cause of morbidity throughout the tropics; however, accurate population-based estimates of mortality rates are not available.We established the Enhanced Fatal Acute Febrile Illness Surveillance System (EFASS) to estimate dengue mortality rates in Puerto Rico. Healthcare professionals submitted serum and tissue specimens from patients who died from a dengue-like acute febrile illness, and death certificates were reviewed to identify additional cases. Specimens were tested for markers of dengue virus (DENV) infection by molecular, immunologic, and immunohistochemical methods, and were also tested for West Nile virus, Leptospira spp., and other pathogens based on histopathologic findings. Medical records were reviewed and clinical data abstracted. A total of 311 deaths were identified, of which 58 (19%) were DENV laboratory-positive. Dengue mortality rates were 1.05 per 100,000 population in 2010, 0.16 in 2011 and 0.36 in 2012. Dengue mortality was highest among adults 19-64 years and seniors ≥65 years (1.17 and 1.66 deaths per 100,000, respectively). Other pathogens identified included 34 Leptospira spp. cases and one case of Burkholderia pseudomallei and Neisseria meningitidis.EFASS showed that dengue mortality rates among adults were higher than reported for influenza, and identified a leptospirosis outbreak and index cases of melioidosis and meningitis.

Three clusters of organ transplant–associated lymphocytic choriomeningitis virus (LCMV) transmissions have been identified in the United States; 9 of 10 recipients died. In February 2011, we identified a fourth cluster of organ transplant–associated LCMV infections. Diabetic ketoacidosis developed in the organ donor in December 2010; she died with generalized brain edema after a short hospitalization. Both kidneys, liver, and lung were transplanted to 4 recipients; in all 4, severe posttransplant illness developed; 2 recipients died. Through multiple diagnostic methods, we identified LCMV infection in all persons, including in at least 1 sample from the donor and 4 recipients by reverse transcription PCR, and sequences of a 396-bp fragment of the large segment of the virus from all 5 persons were identical. In this cluster, all recipients developed severe illness, but 2 survived. LCMV infection should be considered as a possible cause of severe posttransplant illness.

We compared the prevalence of 8 polymorphisms in the tumor necrosis factor and mannose-binding lectin genes among 105 children and young adults with fatal influenza with US population estimates and determined in subanalyses whether these polymorphisms were associated with sudden death and bacterial co-infection among persons with fatal influenza. No differences were observed in genotype prevalence or minor allele frequencies between persons with fatal influenza and the reference sample. Fatal cases with low-producing MBL2 genotypes had a 7-fold increased risk for invasive methicillin-resistant Staphylococcus aureus (MRSA) co-infection compared with fatal cases with high- and intermediate-producing MBL2 genotypes (odds ratio 7.1, 95% confidence interval 1.6–32.1). Limited analysis of 2 genes important to the innate immune response found no association between genetic variants and fatal influenza infection. Among children and young adults who died of influenza, low-producing MBL2 genotypes may have increased risk for MRSA co-infection.

We evaluated pandemic influenza A (H1N1) 2009 virus isolates and respiratory tissues collected at autopsy by electron microscopy. Many morphologic characteristics were similar to those previously described for influenza virus. One of the distinctive features was dense tubular structures in the nuclei of infected cells.

The Phylum Microsporidia comprises >1,200 species, only 15 of which are known to infect humans, including the genera Trachipleistophora, Pleistophora, and Brachiola. We report an infection by Tubulinosema sp. in an immunosuppressed patient.

Encephalitis is a major cause of death worldwide. Although >100 pathogens have been identified as causative agents, the pathogen is not determined for up to 75% of cases. This diagnostic failure impedes effective treatment and underscores the need for better tools and new approaches for detecting novel pathogens or determining new manifestations of known pathogens. Although astroviruses are commonly associated with gastroenteritis, they have not been associated with central nervous system disease. Using unbiased pyrosequencing, we detected an astrovirus as the causative agent for encephalitis in a 15-year-old boy with agammaglobulinemia; several laboratories had failed to identify the agent. Our findings expand the spectrum of causative agents associated with encephalitis and highlight unbiased molecular technology as a valuable tool for differential diagnosis of unexplained disease.

A nosocomial outbreak of disease involving 5 patients, 4 of whom died, occurred in South Africa during September–October 2008. The first patient had been transferred from Zambia to South Africa for medical management. Three cases involved secondary spread of infection from the first patient, and 1 was a tertiary infection. A novel arenavirus was identified. The source of the first patient’s infection remains undetermined.

The only variant Creutzfeldt-Jakob disease (vCJD) patient identified in the United States died in 2004, and the diagnosis was confirmed by analysis of autopsy tissue. The patient likely acquired the disease while growing up in Great Britain before immigrating to the United States in 1992. Additional vCJD patients continue to be identified outside the United Kingdom, including 2 more patients in Ireland, and 1 patient each in Japan, Portugal, Saudi Arabia, Spain, and the Netherlands. The reports of bloodborne transmission of vCJD in 2 patients, 1 of whom was heterozygous for methionine and valine at polymorphic codon 129, add to the uncertainty about the future of the vCJD outbreak.

West Nile virus (WNV) causes epidemics of febrile illness, meningitis, encephalitis, and flaccid paralysis. Since it was first detected in New York City in 1999, and through 2004, 16,000 WNV disease cases have been reported in the United States. Over the past 5 years, research on WNV disease has expanded rapidly. This review highlights new information regarding the virology, clinical manifestations, and pathology of WNV disease, which will provide a new platform for further research into diagnosis, treatment, and possible prevention of WNV through vaccination.

We identified West Nile virus (WNV) RNA in skin, fat, muscle, tendon, and bone marrow from a deceased donor associated with WNV transmission through solid organ transplantation. WNV could not be cultured from the RNA-positive tissues. Further studies are needed to determine if WNV can be transmitted from postmortem tissues.

We report a fatal case of rickettsiosis in a woman from the United States living in Kenya, who had a history of tick exposure. Immunohistochemical staining of skin, kidney, and liver demonstrated spotted fever group rickettsiae. The clinical findings, severity, and fatal outcome are most consistent with Rickettsia conorii infection.

During May and June 2003, the first cluster of human monkeypox cases in the United States was reported. Most patients with this febrile vesicular rash illness presumably acquired the infection from prairie dogs. Monkeypox virus was demonstrated by using polymerase chain reaction in two prairie dogs in which pathologic studies showed necrotizing bronchopneumonia, conjunctivitis, and tongue ulceration. Immunohistochemical assays for orthopoxviruses demonstrated abundant viral antigens in surface epithelial cells of lesions in conjunctiva and tongue, with less amounts in adjacent macrophages, fibroblasts, and connective tissues. Viral antigens in the lung were abundant in bronchial epithelial cells, macrophages, and fibroblasts. Virus isolation and electron microscopy demonstrated active viral replication in lungs and tongue. These findings indicate that both respiratory and direct mucocutaneous exposures are potentially important routes of transmission of monkeypox virus between rodents and to humans. Prairie dogs offer insights into transmission, pathogenesis, and new vaccine and treatment trials because they are susceptible to severe monkeypox infection.

Leptospirosis is a reemerging infectious disease in California. Leptospirosis is the most widespread zoonosis throughout the world, though it is infrequently diagnosed in the continental United States. From 1982 to 2001, most reported California cases occurred in previously healthy young adult white men after recreational exposures to contaminated freshwater. We report five recent cases of human leptospirosis acquired in California, including the first documented common-source outbreak of human leptospirosis acquired in this state, and describe the subsequent environmental investigation. Salient features in the California cases include high fever with uniform renal impairment and mild hepatitis. Because leptospirosis can progress rapidly if untreated, this reemerging infection deserves consideration in febrile patients with a history of recreational freshwater exposure, even in states with a low reported incidence of infection.

We describe a case of Q fever endocarditis in an HIV-infected patient. The case was treated successfully with valvular replacement and a combination of doxycycline and hydroxychloroquine. We review the current literature on Q fever endocarditis, with an emphasis on the co-infection of HIV and Coxiella burnetii.

Severe acute respiratory syndrome (SARS) was first described during a 2002–2003 global outbreak of severe pneumonia associated with human deaths and person-to-person disease transmission. The etiologic agent was initially identified as a coronavirus by thin-section electron microscopic examination of a virus isolate. Virions were spherical, 78 nm in mean diameter, and composed of a helical nucleocapsid within an envelope with surface projections. Herein, we show that infection with the SARS-associated coronavirus resulted in distinct ultrastructural features: double-membrane vesicles, nucleocapsid inclusions, and large granular areas of cytoplasm. These three structures and the coronavirus particles were shown to be positive for viral proteins and RNA by using ultrastructural immunogold and in situ hybridization assays. In addition, ultrastructural examination of a bronchiolar lavage specimen from a SARS patient showed numerous coronavirus-infected cells with features similar to those in infected culture cells. Electron microscopic studies were critical in identifying the etiologic agent of the SARS outbreak and in guiding subsequent laboratory and epidemiologic investigations.