Cynthia S. Goldsmith

Rickettsia parkeri: A Newly Recognized Cause of Spotted Fever Rickettsiosis in the United States

Ticks, including many that bite humans, are hosts to several obligate intracellular bacteria in the spotted fever group (SFG) of the genus Rickettsia. Only Rickettsia rickettsii, the agent of Rocky Mountain spotted fever, has been definitively associated with disease in humans in the United States. Herein we describe disease in a human caused by Rickettsia parkeri, an SFG rickettsia first identified >60 years ago in Gulf Coast ticks (Amblyomma maculatum) collected from the southern United States. Confirmation of the infection was accomplished using serological testing, immunohistochemical staining, cell culture isolation, and molecular methods. Application of specific laboratory assays to clinical specimens obtained from patients with febrile, eschar-associated illnesses following a tick bite may identify additional cases of R. parkeri rickettsiosis and possibly other novel SFG rickettsioses in the United States.

A New Phlebovirus Associated with Severe Febrile Illness in Missouri

Two men from northwestern Missouri independently presented to a medical facility with fever, fatigue, diarrhea, thrombocytopenia, and leukopenia, and both had been bitten by ticks 5 to 7 days before the onset of illness. Ehrlichia chaffeensis was suspected as the causal agent but was not found on serologic analysis, polymerase-chain-reaction (PCR) assay, or cell culture. Electron microscopy revealed viruses consistent with members of the Bunyaviridae family. Next-generation sequencing and phylogenetic analysis identified the viruses as novel members of the phlebovirus genus. Although Kochs postulates have not been completely fulfilled, we believe that this phlebovirus, which is novel in the Americas, is the cause of this clinical syndrome.

2009 Pandemic Influenza A (H1N1): Pathology and Pathogenesis of 100 Fatal Cases in the United States

In the spring of 2009, a novel influenza A (H1N1) virus emerged in North America and spread worldwide to cause the first influenza pandemic since 1968. During the first 4 months, over 500 deaths in the United States had been associated with confirmed 2009 pandemic influenza A (H1N1) [2009 H1N1] virus infection. Pathological evaluation of respiratory specimens from initial influenza-associated deaths suggested marked differences in viral tropism and tissue damage compared with seasonal influenza and prompted further investigation. Available autopsy tissue samples were obtained from 100 US deaths with laboratory-confirmed 2009 H1N1 virus infection. Demographic and clinical data of these case-patients were collected, and the tissues were evaluated by multiple laboratory methods, including histopathological evaluation, special stains, molecular and immunohistochemical assays, viral culture, and electron microscopy. The most prominent histopathological feature observed was diffuse alveolar damage in the lung in all case-patients examined. Alveolar lining cells, including type I and type II pneumocytes, were the primary infected cells. Bacterial co-infections were identified in >25% of the case-patients. Viral pneumonia and immunolocalization of viral antigen in association with diffuse alveolar damage are prominent features of infection with 2009 pandemic influenza A (H1N1) virus. Underlying medical conditions and bacterial co-infections contributed to the fatal outcome of this infection. More studies are needed to understand the multifactorial pathogenesis of this infection.

Nipah Virus Infection: Pathology and Pathogenesis of an Emerging Paramyxoviral Zoonosis

In 1998, an outbreak of acute encephalitis with high mortality rates among pig handlers in Malaysia led to the discovery of a novel paramyxovirus named Nipah virus. A multidisciplinary investigation that included epidemiology, microbiology, molecular biology, and pathology was pivotal in the discovery of this new human infection. Clinical and autopsy findings were derived from a series of 32 fatal human cases of Nipah virus infection. Diagnosis was established in all cases by a combination of immunohistochemistry (IHC) and serology. Routine histological stains, IHC, and electron microscopy were used to examine autopsy tissues. The main histopathological findings included a systemic vasculitis with extensive thrombosis and parenchymal necrosis, particularly in the central nervous system. Endothelial cell damage, necrosis, and syncytial giant cell formation were seen in affected vessels. Characteristic viral inclusions were seen by light and electron microscopy. IHC analysis showed widespread presence of Nipah virus antigens in endothelial and smooth muscle cells of blood vessels. Abundant viral antigens were also seen in various parenchymal cells, particularly in neurons. Infection of endothelial cells and neurons as well as vasculitis and thrombosis seem to be critical to the pathogenesis of this new human disease.

Highly Pathogenic Avian Influenza H5N1 Viruses Elicit an Attenuated Type I Interferon Response in Polarized Human Bronchial Epithelial Cells

ABSTRACT The unparalleled spread of highly pathogenic avian influenza A (HPAI) H5N1 viruses has resulted in devastating outbreaks in domestic poultry and sporadic human infections with a high fatality rate. To better understand the mechanism(s) of H5N1 virus pathogenesis and host responses in humans, we utilized a polarized human bronchial epithelial cell model that expresses both avian alpha-2,3- and human alpha-2,6-linked sialic acid receptors on the apical surface and supports productive replication of both H5N1 and H3N2 viruses. Using this model, we compared the abilities of selected 2004 HPAI H5N1 viruses isolated from humans and a recent human H3N2 virus to trigger the type I interferon (IFN) response. H5N1 viruses elicited significantly less IFN regulatory factor 3 (IRF3) nuclear translocation, as well as delayed and reduced production of IFN-β compared with the H3N2 virus. Furthermore, phosphorylation of Stat2 and induction of IFN-stimulated genes (ISGs), such as MX1, ISG15, IRF7, and retinoic acid-inducible gene I, were substantially delayed and reduced in cells infected with H5N1 viruses. We also observed that the highly virulent H5N1 virus replicated more efficiently and induced a weaker IFN response than the H5N1 virus that exhibited low virulence in mammals in an earlier study. Our data suggest that the H5N1 viruses tested, especially the virus with the high-pathogenicity phenotype, possess greater capability to attenuate the type I IFN response than the human H3N2 virus. The attenuation of this critical host innate immune defense may contribute to the virulence of H5N1 viruses observed in humans.

Monkeypox transmission and pathogenesis in prairie dogs.

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.

Pathology of congenital Zika syndrome in Brazil: a case series

BACKGROUND Zika virus is an arthropod-borne virus that is a member of the family Flaviviridae transmitted mainly by mosquitoes of the genus Aedes. Although usually asymptomatic, infection can result in a mild and self-limiting illness characterised by fever, rash, arthralgia, and conjunctivitis. An increase in the number of children born with microcephaly was noted in 2015 in regions of Brazil with high transmission of Zika virus. More recently, evidence has been accumulating supporting a link between Zika virus and microcephaly. Here, we describe findings from three fatal cases and two spontaneous abortions associated with Zika virus infection. METHODS In this case series, formalin-fixed paraffin-embedded tissue samples from five cases, including two newborn babies with microcephaly and severe arthrogryposis who died shortly after birth, one 2-month-old baby, and two placentas from spontaneous abortions, from Brazil were submitted to the Infectious Diseases Pathology Branch at the US Centers for Disease Control and Prevention (Atlanta, GA, USA) between December, 2015, and March, 2016. Specimens were assessed by histopathological examination, immunohistochemical assays using a mouse anti-Zika virus antibody, and RT-PCR assays targeting the NS5 and envelope genes. Amplicons of RT-PCR positive cases were sequenced for characterisation of strains. FINDINGS Viral antigens were localised to glial cells and neurons and associated with microcalcifications in all three fatal cases with microcephaly. Antigens were also seen in chorionic villi of one of the first trimester placentas. Tissues from all five cases were positive for Zika virus RNA by RT-PCR, and sequence analyses showed highest identities with Zika virus strains isolated from Brazil during 2015. INTERPRETATION These findings provide strong evidence of a link between Zika virus infection and different congenital central nervous system malformations, including microcephaly as well as arthrogryposis and spontaneous abortions. FUNDING None.

Ultrastructural Characterization of SARS Coronavirus

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.

Modern Uses of Electron Microscopy for Detection of Viruses

SUMMARY Electron microscopy, considered by some to be an old technique, is still on the forefront of both clinical viral diagnoses and viral ultrastructure and pathogenesis studies. In the diagnostic setting, it is particularly valuable in the surveillance of emerging diseases and potential bioterrorism viruses. In the research arena, modalities such as immunoelectron microscopy, cryo-electron microscopy, and electron tomography have demonstrated how viral structural components fit together, attach to cells, assimilate during replication, and associate with the cellular machinery during replication and egression. These studies provide information for treatment and vaccine strategies.

Ultrastructural characteristics of Sin Nombre virus, causative agent of hantavirus pulmonary syndrome.

SummaryA previously unrecognized disease, hantavirus pulmonary syndrome, was described following an outbreak of severe, often lethal, pulmonary illness in the southwestern United States in May–June, 1993. We have now studied the morphologic features of the causative agent, Sin Nombre virus (SNV), by thin section electron microscopy and immunoelectron microscopy of infected Vero E6 cells. SNV virions were roughly spherical and had a mean diameter of 112 nm. They had a rather dense envelope and closely apposed fine surface projections, 7 nm in length. Filamentous nucleocapsids were present within virions. Viral inclusion bodies were present in the cytoplasm of infected cells; these appeared granular or filamentous, depending on the plane of section. All of these characteristics were similar to published descriptions of other hantaviruses; however, unlike all other hantaviruses and virtually all other member viruses of the familyBunyaviridae which bud upon smooth intracytoplasmic membranes, SNV budding occurred almost entirely upon the plasma membrane of infected cells. Virus budding was associated with the formation of long 28 nm diameter tubular projections. Occasional elongated 47 nm diameter virus-like particles were seen to bud upon intracytoplasmic membranes. As shown by immunoelectron microscopy, viral antigens were localized over virions, inclusions, and tubular projections associated with virion morphogenesis.