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A 38-year-old patient with systemic lupus erythematosus presented with pulmonary infiltrates and hypoxemia for several months following immunodepleting autologous hematopoietic stem cell transplantation. She was treated for influenza, which was isolated repeatedly from ororpharynx and bronchoalveolar lavage fluids, and later empirically for lupus pneumonitis, but expired 6 months after transplant. Autopsy findings failed to show influenza in the lungs or lupus pneumonitis. A novel generic PCR-based assay using degenerate primers identified human coronavirus HKU1 RNA in bronchoalveolar lavage fluid at autopsy. Coronavirus was confirmed by virus-specific PCRs of lung tissue at autopsy. Electron microscopy showed viral particles consistent with coronavirus HKU1 in lung tissue both at autopsy and from a previous biopsy. While human coronavirus HKU1 infection is not usually severe, in highly immunocompromised patients, it can be associated with fatal pneumonia.

A cytopathic virus was isolated using Madin-Darby bovine kidney (MDBK) cells from lung tissue of alpaca that died of a severe respiratory infection. To identify the virus, the infected cell culture supernatant was enriched for virus particles and a generic, PCR-based method was used to amplify potential viral sequences. Genomic sequence data of the alpaca isolate was obtained and compared with sequences of known viruses. The new alpaca virus sequence was most similar to recently designated Enterovirus species F, previously bovine enterovirus (BEVs), viruses that are globally prevalent in cattle, although they appear not to cause significant disease. Because bovine enteroviruses have not been previously reported in U.S. alpaca, we suspect that this type of infection is fairly rare, and in this case appeared not to spread beyond the original outbreak. The capsid sequence of the detected virus had greatest homology to Enterovirus F type 1 (indicating that the virus should be considered a member of serotype 1), but the virus had greater homology in 2A protease sequence to type 3, suggesting that it may have been a recombinant. Identifying pathogens that infect a new host species for the first time can be challenging. As the disease in a new host species may be quite different from that in the original or natural host, the pathogen may not be suspected based on the clinical presentation, delaying diagnosis. Although this virus replicated in MDBK cells, existing standard culture and molecular methods could not identify it. In this case, a highly sensitive generic PCR-based pathogen-detection method was used to identify this pathogen.

Largemouth bass virus (LMBV), a recently discovered iridovirus found in the eastern United States, is usually detected by isolation in cell culture. Although LMBV will replicate in several cell lines, optimal cell culture methods for the detection of this virus have not been determined. We tested inoculation method, adsorption time, incubation temperature, and various cell lines to determine the conditions that would provide the most sensitive cell culture assay for LMBV. The optimal inoculation procedure tested was to remove the culture medium from the culture well before the addition of the inoculum, and the optimal adsorption procedure tested was to allow the virus to adsorb for 40 min while the plates were on an orbital shaker. Following inoculation, incubation at 30°C resulted in a higher number of viral plaques than incubation at 25°C or 32°C. Four cell lines (bluegill fry (BF‐2), fathead minnow (FHM), epithelioma papulosum cyprini, and channel catfish ovary cells) inoculated with LMBV had similar susceptibility to infection. Similar percentages of LMBV‐positive samples were detected in BF‐2 and FHM cell cultures inoculated with homogenized organ samples from largemouth bass Micropterus salmoides; however, the use of two cell lines increased the number of infected samples discovered. A blind passage also increased the number of positive samples detected in cell culture. Subcultivation to confirm virus‐positive samples was useful for reducing false‐positive results.

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