Charles D. Humphrey

Characterization of human metapneumoviruses isolated from patients in North America.

Abstract Human metapneumovirus (HMPV) was recently identified in The Netherlands and was linked to acute respiratory tract illness. In this study, 11 isolates from 10 patients with respiratory disease from Quebec, Canada, were tested by a reverse-transcriptase polymerase chain reaction based on the fusion protein gene. Identified sequences were consistent with HMPV. The patients were 2 months to 87 years of age (median age, 58 years) and presented with acute respiratory tract illness during the winter season. Sequence studies of the nucleocapsid, fusion, and polymerase genes identified 2 main lineages of HMPV and cocirculation of both lineages during the same year. These findings support a previous finding that HMPV is a human respiratory pathogen that merits further study.

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.

Aetiological Agent of Enterically Transmitted Non-A, Non-B Hepatitis

Virus-like particles (VLPs) with a mean diameter of 32 nm were recovered from the stools of three acute phase cases of enterically transmitted non-A, non-B hepatitis (ET-NANBH) occurring in the Soviet Union, North Africa and North America. VLPs from two of these cases were studied in detail and were shown to react specifically with antibody in acute phase sera obtained from other cases of ET-NANBH in Asia, the Soviet Union, North Africa and North America. Partially purified VLPs were found to sediment at 183S in sucrose gradients and to cross-react with antibody in acute phase sera from geographically isolated cases of ET-NANBH. The latter virus preparations were also used to document the seroconversion of experimentally ET-NANBH-infected cynomolgus macaques to 32 nm VLPs. Our findings indicate that one virus or class of viruses is responsible for the majority of ET-NANBH.

Parkville virus : A novel genetic variant of human calicivirus in the Sapporo virus clade, associated with an outbreak of gastroenteritis in adults

This report describes the characterization of Parkville virus, the etiologic agent of an outbreak of foodborne gastroenteritis, that has the morphology of a calicivirus and genetic properties that distinguish it from previously identified strains in the Sapporo/Manchester virus clade. Sequence analysis of the Parkville virus genome showed it contained the RNA‐dependent RNA polymerase motifs GLPSG and YGDD characteristic of members of the family Caliciviridae with an organization identical to that reported for the Manchester virus where the capsid region of the polyprotein is fused to the RNA polymerase. Parkville virus however, demonstrates considerable sequence divergence from both the Manchester and Sapporo caliciviruses, providing the first indications that genetic diversity exists within caliciviruses of this previously homogeneous clade. On the basis of recent advances in the genetic characterization of members of the family Caliciviridae, we propose a new interim phylogenetic classification system in which Parkville virus would be included with Manchester and Sapporo virus as a separate group distinct from the small round‐structured viruses (Norwalk‐like viruses) that also cause diarrhea in humans. J. Med. Virol. 52:173–178, 1997.

Descriptive epidemiology of fatal respiratory outbreaks and detection of a human-related metapneumovirus in wild chimpanzees (Pan troglodytes) at Mahale Mountains National Park, Western Tanzania

Over the past several years, acute and fatal respiratory illnesses have occurred in the habituated group of wild chimpanzees at the Mahale Mountains National Park, Tanzania. Common respiratory viruses, such as measles and influenza, have been considered possible causative agents; however, neither of these viruses had been detected. During the fatal respiratory illnesses in 2003, 2005 and 2006, regular observations on affected individuals were recorded. Cause‐specific morbidity rates were 98.3, 52.4 and 33.8%, respectively. Mortality rates were 6.9, 3.2 and 4.6%; all deaths were observed in infants 2 months–2 years 9 months of age. Nine other chimpanzees have not been seen since the 2006 outbreak and are presumed dead; hence, morbidity and mortality rates for 2006 may be as high as 47.7 and 18.5%, respectively. During the 2005 and 2006 outbreaks, 12 fecal samples were collected from affected and nonaffected chimpanzees and analyzed for causative agents. Analysis of fecal samples from 2005 suggests the presence of paramyxovirus, and in 2006 a human‐related metapneumovirus was detected and identified in an affected chimpanzee whose infant died during the outbreak. Our findings provide preliminary evidence that the causative agent associated with these illnesses is viral and contagious, possibly of human origin; and that, possibly more than one agent may be circulating in the population. We recommend that baseline health data be acquired and food wadge and fecal samples be obtained and bio‐banked as early as possible when attempting to habituate new groups of chimpanzees or other great apes. For already habituated populations, disease prevention strategies, ongoing health monitoring programs and reports of diagnostic findings should be an integral part of managing these populations. In addition, descriptive epidemiology should be a major component of disease outbreak investigations. Am. J. Primatol. 70: 755–765, 2008.

Characterization of Toronto virus capsid protein expressed in baculovirus

SummaryToronto virus (TV), previously called “minireovirus,” a human calicivirus classified as genogroup 2 and phylogenetic type P2-A, was originally described in association with diarrhea in children. The second open reading frame, encoding the capsid protein of TV24, was expressed in a baculovirus recombinant. The recombinant baculovirus produced a protein (rTV) with an apparent molecular mass of 58 kDa that self-assembled into virus-like particles ∼ 30 nm in diameter with a density of 1.29 g/ml. Antigenic and immunogenic characteristics of these particles were determined by protein immunoblot, immunoprecipitation, and enzyme immunoassay. Seroconversion to the rTV protein was detected in 6 of 8 (75%) patients from a recent outbreak of gastroenteritis associated with a virus of similar phylogenetic type. These results confirm and extend the previous reports of the expression of the Norwalk and Mexico virus capsid proteins.

Detection of viral agents in fecal specimens of monkeys with diarrhea

Background  Diarrheal disease is a major cause of morbidity and mortality in humans and animals, including non human primates. While the diagnostics for gastrointestinal bacterial and parasitic pathogens and their etiological role in disease are well established, little is known about the epidemiology, prevalence and role of viral agents in diarrheal illness among monkeys.

Characterization of Capsid Genes, Expressed in the Baculovirus System, of Three New Genetically Distinct Strains of “Norwalk-Like Viruses”

ABSTRACT “Norwalk-like viruses” (NLVs), members of a newly defined genus of the family Caliciviridae, are the most common agents of outbreaks of gastroenteritis in the United States. Two features of NLVs have hindered the development of simple methods for detection and determination of serotype: their genetic diversity and their inability to grow in cell culture. To assess the immune responses of patients involved in outbreaks of gastroenteritis resulting from infection with NLVs, we previously used recombinant-expressed capsid antigens representing four different genetic clusters, but this panel proved insufficient for detection of an immune response in many patients. To extend and further refine this panel, we expressed in baculovirus the capsid genes of three additional genetically distinct viruses, Burwash Landing virus (BLV), White River virus (WRV), and Florida virus. All three expressed proteins assembled into virus-like particles (VLPs) that contained a full-length 64-kDa protein, but both the BLV and WRV VLPs also contained a 58-kDa protein that resulted from deletion of 39 amino acids at the amino terminus. The purified VLPs were used to measure the immune responses in 403 patients involved in 37 outbreaks of acute gastroenteritis. A majority of patients demonstrated a fourfold rise in the titer of immunoglobulin G to the antigen homologous to the outbreak strain, but most seroconverted in response to other genetically distinct antigens as well, suggesting no clear pattern of type-specific immune response. Further study of the antigenicity of the NLVs by use of VLPs should allow us to design new detection systems with either broader reactivity or better specificity and to define the optimum panel of antigens required for routine screening of patient sera.

Outbreaks of gastroenteritis in elderly nursing homes and retirement facilities associated with human caliciviruses

Eleven outbreaks of acute gastroenteritis, eight of which were in nursing homes or retirement facilities, were reported in Virginia during the winter of 1993–1994. Serum samples (four outbreaks) and stool samples (two outbreaks) from involved people were tested for human calicivirus (HuCV) infection by enzyme immune assays (EIAs) using recombinant Norwalk virus (rNV) and Mexico virus (rMX) capsid antigens and reverse transcription‐polymerase chain reaction (RT‐PCR). Of the 31 pairs of acute and convalescent serum specimens tested, 24 had a fourfold or more titer increase to rMX and 4 responded to rNV. In all four outbreaks, the geometric mean titers (GMTs) against rMX were significantly higher than those against rNV in the covalescent, but not in the acute phase of illness. The antibody response to rMX among these patients was also higher than to rNV (summary mean 32‐fold increase vs. 0.7‐fold increase, respectively, P < .001). Antigen was detected in 5 of 21 stool specimens tested by the rMX EIA, RNA in 12 of 17 stool specimens tested by RT‐PCR, and small round structured virus (SRSV) particles in 12 of 21 by electron microscopy (EM); none were positive by the rNV EIA. Sequence analysis of the RT‐PCR‐amplified products from the viral RNA polymerase region revealed 92–93% amino acid identity with Snow Mountain agent (SMA), 86% with MX, 58–59% with NV, and 31–32% with Sapporo HuCV, suggesting that these viruses belong to the SMA HuCV genogroup.

Development of plaque assays for adenoviruses 40 and 41

Enteric adenoviruses, important agents of infantile gastroenteritis, are difficult to culture with low titers and limited CPE. Consequently, few plaque assays have been reported and none are used routinely by investigators who may need reproducible quantitative assays for these viruses. CPE in A549 cells (an epithelial lung carcinoma cell line) was induced by isolates of human adenovirus (HAdV) serotypes 40 or 41 that were obtained by prior limited passage in primary cynmolgous monkey kidney (pCMK), human embryonic kidney (HEK), and Graham 293 cells. CPE with HAdV 40 (Dugan strain) and HAdV 41 (Tak strain) inoculated in A549 cells was also observed. Monolayers of A549 cells were inoculated with a low multiplicity of infection (MOI) of the archived stock isolates and harvested at days 10-14 with full CPE. Subsequent passages were harvested in as few as 7 days with 100% CPE to prepare virus stocks for plaque assay. Large individual plaques under agarose overlay were picked prior to staining and clonal stocks prepared. Titers of final stock preparations after six to eight passages in A549 cells were in the range of 5 x 10(7)-1 x 10(8)PFU/ml, which provides adequate virus for quantitative recovery studies. The particle to infectivity (P:I) ratios of the early passages of virus stocks were in the range reported previously. The ratio of non-infectious to infectious particles decreased with successive passages of HAdVs 40 and 41 in A549 cells. The specificity of the assay was confirmed by neutralization of plaques with type-specific antisera. Furthermore, sequence analysis of the HAdVs 40 and 41 plaque forming stocks ruled out contamination with any other HAdVs. The plaque assay developed will be useful for evaluation of virus recovery methods from water, food or other environmental matrices, as well as determination of the efficacy of water treatment techniques for inactivation of these viruses.