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Twenty autopsy cases with 2009 pandemic influenza A (2009 H1N1) virus infection, performed between August 2009 and February 2010, were histopathologically analyzed. Hematoxylin-eosin staining, immunohistochemistry for type A influenza nucleoprotein antigen, and real-time reverse transcription-PCR assay for viral RNA were performed on formalin-fixed and paraffin-embedded specimens. In addition, the D222G amino acid substitution in influenza virus hemagglutinin, which binds to specific cell receptors, was analyzed in formalin-fixed and paraffin-embedded trachea and lung sections by direct sequencing of PCR-amplified products. There were several histopathological patterns in the lung according to the most remarkable findings in each case: acute diffuse alveolar damage (DAD) with a hyaline membrane (four cases), organized DAD (one case), acute massive intra-alveolar edema with variable degrees of hemorrhage (three cases), neutrophilic bronchopneumonia (five cases) and tracheobronchitis with limited histopathological changes in alveoli (four cases). In two cases, the main findings were due to preexisting disease. Influenza virus antigen was only detected in the respiratory tract in 10 cases by immunohistochemistry. The antigen was detected in type II pneumocytes (three cases) in the epithelial cells of the trachea, bronchi and glands (six cases), and in the epithelial cells in both of the above (one case). The four cases with acute DAD presented with antigen-positive type II pneumocytes. In one case, the D222G substitution was detected in the lung as a major sequence, although 222D was prominent in the trachea, suggesting that selection of the viral clones occurred in the respiratory tract. In five cases, the pathogenesis of 2009 H1N1 was confirmed to be viral infection in pneumocytes, which caused severe alveolar damage and fatal viral pneumonia. Further studies on both host and viral factors in autopsy or biopsy materials will be essential to elucidate the other pathogenic factors involved in influenza virus infection.

The AT-tailing method is a labelling technique that utilises oligo(dA-dT)-dependent signal amplification. In this study, a new immunohistochemical application of the immunoAT method was developed. This method uses an oligo(dA-dT)-conjugated primary antibody (direct immunoAT method) or an oligo(dA-dT)-conjugated secondary antibody (indirect immunoAT method). Fifteen-base oligo(dA-dT)-conjugated antibodies (IgG-ATs) were prepared in advance by conjugating maleimide-activated oligo(dA-dT) to IgG via free sulfhydryl residues that had been introduced on the surface of IgG using Traut's reagent. Following the reaction with the target antigen and the IgG-AT, oligo(dA-dT) was elongated by DeltaTth DNA polymerase in the presence of dATP, dTTP and biotinylated dUTP, consequently labelling the antigen-antibody complex with a large amount of biotin. To initially evaluate the immunoAT method, the presence or absence of prion protein (PrP(sc)) was determined in formalin-fixed and paraffin-embedded sections of the medulla oblongata of cattle which had been under active surveillance for bovine spongiform encephalopathy. Sections were examined using direct and indirect immunoAT methods and the EnVision+ system (Dako) under conditions that were identical except for the differing IgG-AT and AT-tailing methods. PrP(sc) detection was consistent using all three methods. The clearest signals were obtained using the indirect immunoAT method, suggesting significant potential for this method., (Copyright 2010 Elsevier B.V. All rights reserved.)

An autopsy of a patient in Japan with coronavirus disease indicated pneumonia lung pathology, manifested as diffuse alveolar damage. We detected severe acute respiratory syndrome coronavirus 2 antigen in alveolar epithelial cells and macrophages. Coronavirus disease is essentially a lower respiratory tract disease characterized by direct viral injury of alveolar epithelial cells. [ABSTRACT FROM AUTHOR]

The aim of this study was to establish a reliable method of virus detection for the diagnosis of critical enterovirus infections such as acute infective encephalitis, encephalomyelitis and myocarditis. Because histopathological and immunohistochemical analyses of paraffin-embedded tissues play an important role in recognizing infectious agents in tissue samples, six in-house polyclonal antibodies raised against three representative enteroviruses using an indirect immunofluorescence assay and immunohistochemistry were examined. This panel of polyclonal antibodies recognized three serotypes of enterovirus. Two of the polyclonal antibodies were raised against denatured virus particles from enterovirus A71, one was raised against the recombinant VP1 protein of coxsackievirus B3, and the other for poliovirus type 1 were raised against denatured virus particles, the recombinant VP1 protein and peptide 2C. Western blot analysis revealed that each of these antibodies recognized the corresponding viral antigen and none cross-reacted with non-enteroviruses within the family Picornaviridae. However, all cross-reacted to some extent with the antigens derived from other serotypes of enterovirus. Indirect immunofluorescence assay and immunohistochemistry revealed that the virus capsid and non-structural proteins were localized in the cytoplasm of affected culture cells, and skeletal muscles and neurons in neonatal mice experimentally-infected with human enterovirus. The antibodies also recognized antigens derived from recent clinical isolates of enterovirus A71, coxsackievirus B3 and poliovirus. In addition, immunohistochemistry revealed that representative antibodies tested showed the same recognition pattern according to each serotype. Thus, the panel of in-house anti-enterovirus polyclonal antibodies described herein will be an important tool for the screening and pathological diagnosis for enterovirus infections, and may be useful for the classification of different enterovirus serotypes, including coxsackieviruses A and B, echoviruses, enterovirus A71 and poliovirus. [ABSTRACT FROM AUTHOR]

Two Japanese men, 65 and 69 years old, developed rabies in Japan around 2–3 months after dog-bite exposure in the Philippines. Laboratory diagnosis of rabies was made following the detection of rabies virus genome on reverse transcription–polymerase chain reaction from saliva, and on immunohistochemistry of a nuchal skin punch biopsy in one case. The patients died 9 and 19 days after clinical onset. At autopsy, no macroscopic changes in the CNS were observed. Histopathology indicated that eosinophilic and cytoplasmic inclusion bodies, Negri bodies, were seen in neuronal cells of the CNS. Inflammatory cell reactions were scarce, and no apoptosis in the CNS was detected. Immunohistochemistry demonstrated that rabies virus nucleoprotein (N) and phosphoprotein (P) were disseminated to all neural tissues and cells in the body with a similar pattern in both cases. Interestingly, there were no differences of localization between N and P antigen in the brain, but the N antigen was located at the peripheral nerve sheaths and the P antigen was localized in axons. These data indicate that rabies virus dissemination in all neural tissues causes disease development and death. Immunohistochemistry for rabies is a powerful tool to understand the pathogenesis of rabies. [ABSTRACT FROM AUTHOR]