A blocking test was incorporated into the commercial IDEIA Adenovirus test (DAKO Diagnostics Ltd., Cambridgeshire, UK) to detect false positive results when faecal specimens were tested for adenovirus antigen. Immune rabbit serum raised against pooled adenovirus particles from human faecal specimens, together with the pre-immune serum, was used. Assessment of positive showed that false positives were produced under two different conditions: when results were based on visual determination instead of a cut-off value determined from photometric reading, and when absorbance values were not immediately read at the end of the test. Under the optimum condition for reading and assessment of test results (immediate reading and photometric determination), 11% of 65 adenovirus-positive samples were checked by the blocking ELISA as false positives. The rest of the specimens showed blocking of positive absorbance values by 70 to 98%. ELISA was found to be more sensitive than immune electron microscopy on samples with lower antigen concentration.
Enterovirus A71 (EV-A71) causes hand-foot-and-mouth disease (HFMD), which may be complicated by fatal encephalomyelitis. Although fecal-oral or oral-oral routes are important in person-to-person transmission, how viral shedding and exposure may predispose individuals to infection remains unknown. We investigated person-to-person transmission by using a model of HFMD and encephalomyelitis based on EV-A71 oral infection of 2-week-old hamsters. Animals (index animals) infected with 104 50% cell culture infective doses of virus uniformly developed severe disease four days post-infection (dpi), whereas littermate contacts developed severe disease after six to seven days of exposure to index animals. Virus was detected in oral washes and feces at 3-4 dpi in index animals and at three to eight days after exposure to index animals in littermate contact animals. In a second experiment, non-littermate contact animals exposed for 8 or 12 h to index animals developed the disease six and four days post-exposure, respectively. Tissues from killed index and contact animals, studied by light microscopy, immunohistochemistry and in situ hybridization, exhibited mild inflammatory lesions and/or viral antigens/RNA in the squamous epithelia of the oral cavity, tongue, paws, skin, esophagus, gastric epithelium, salivary glands, lacrimal glands, central nervous system neurons, muscles (skeletal, cardiac and smooth muscles) and liver. Orally shed viruses were probably derived from infected oral mucosa and salivary glands, whereas fecal viruses may have derived from these sites as well as from esophageal and gastric epithelia. Asymptomatic seroconversion in exposed mother hamsters was demonstrated. Our hamster model should be useful in studying person-to-person EV-A71 transmission and how drugs and vaccines may interrupt transmission.
Four nucleic acid extraction protocols were examined for their suitability for extraction of the ssRNA, dsRNA and dsDNA genomes of gastroenteritis viruses, for PCR detection. Protocol (A), employed specimen lysis with guanidinium thiocyanate, extraction with phenol-chloroform-isoamyl alcohol and nucleic acid purification by size-fractionated silica particles. Protocol (B), utilised specimen lysis with guanidinium thiocyanate and nucleic acid purification by silica, followed by phenol-chloroform-isoamyl alcohol extraction. Protocol (C), employed specimen lysis with guanidinium thiocyanate and nucleic acid purification by RNAID glass powder. Protocol (D), employed specimen lysis with sodium dodecyl sulphate, proteinase K digestion and extraction with phenol-chloroform-isoamyl alcohol. Of the four protocols, (B) appeared to be a suitable candidate 'universal' nucleic acid extraction procedure for PCR detection of different viral agents of gastroenteritis in a single nucleic acid extract of a faecal specimen, irrespective of genome composition. Omission of the phenol-chloroform extraction step did not affect negatively the ability of protocol (B) to allow PCR detection of gastroenteritis viruses in faecal specimens. PCR detection of NLVs, astroviruses, rotaviruses and adenoviruses, in single nucleic acid extracts of faecal specimens obtained from the field, confirmed the universality of the modified protocol (B). We propose the modified protocol (B) as a 'universal' nucleic acid extraction procedure, for monoplex PCR detection of gastroenteritis viruses in single nucleic acid extracts of faecal specimens and for development of multiplex PCR for their simultaneous detection.