Pathogenesis of coxsackievirus-B5 acquired from intra-renal porcine islet cell xenografts in diabetic mice.

BACKGROUND: We previously demonstrated the ability of a human isolate of coxsackievirus-B5 (CVB5) to infect productively adult porcine islet cells (PICs) in vitro. PICs infected with CVB5 remain viable, and upon transplantation reversed diabetes in C56BL/6 mice for up to 5 days. METHODS: In the present work, we expanded this graft-to-host xenozoonosis model by examining the long-term functionality of CVB5-infected PIC xenografts in immunosuppressed mice. And, we characterized the pathogenesis of CVB5 infection in mice resulting from directional transmission of the virus from PIC xenografts to surrounding tissues in a mouse model for immunosuppressed human PIC xenograft recipients. RESULTS: Both acutely (12 h) and chronically (72 h) infected PIC xenografts functioned in vivo to reverse diabetes in mice. The efficacy of both infected and un-infected PICs was transient beyond 5 days post-inoculation and the long-term functionality of the grafts was compromised by host-to-graft rejection. CVB5-infected PIC xenografts transmitted infectious virus to immunosuppressed recipient mice resulting in extensive histopathologic changes. The virus replicated in the heart, liver, spleen, kidney, pancreas, brain and skeletal muscle in higher levels in severe-combined immunodeficient (SCID) mice that were directly inoculated with virus when compared to controls. In addition, infectious virus was recovered for up to 22 days after inoculation in SCID mice whereas it was only detected up to Day 4 PI in non-SCID mice. CONCLUSIONS: Immunosuppressed PIC xenograft recipients may be more susceptible to infection with CVB5 which could target the xenograft leading to disseminated infection in the host.

Brachyspira intermedia and Brachyspira pilosicoli are commonly found in older laying flocks in Pennsylvania.

Anaerobic intestinal spirochetes (genus Brachyspira) include several species that are recognized as pathogens of poultry. Surveys undertaken in Europe and Australia have shown that layer and breeder flocks are often colonized by the pathogenic species Brachyspira intermedia and Brachyspira pilosicoli, but similar surveys have not been conducted in the United States. In the current study, DNA was extracted from fecal samples (n=50) collected from each of 21 flocks of laying hens >40 wk of age in Pennsylvania, and this material was tested for B. intermedia and B. pilosicoli using a duplex PCR. Negative samples also were tested using a Brachyspira genus-specific PCR. The consistency of the feces was observed, and manure handling systems and medication histories were recorded. Brachyspira intermedia was detected in 662 (63.1%) samples from 17 (81%) flocks, with a within-flock prevalence of 10%-100%. Brachyspira pilosicoli was detected in 112 (10.7%) samples from 5 flocks (23.8%), with a within-flock prevalence of 8%-82%. Four of the flocks had both pathogenic species present, three had no pathogenic species detected, and two had no Brachyspira species detected. Nine flocks had many fecal samples with a wet appearance and/or a caramel color, and all of these were colonized with one or the other of the two pathogenic species. Nine of 12 flocks with manure that was mainly dry also were colonized. Differences in colonization rates between flocks with or without wet manure were not significant. Colonization with pathogenic Brachyspira species, and particularly B. intermedia, occurs very commonly in layer flocks >40 wk of age in Pennsylvania. The significance of this high rate of colonization requires further investigation.

Prevalent human coxsackie B-5 virus infects porcine islet cells primarily using the coxsackie-adenovirus receptor.

BACKGROUND: We have previously demonstrated that transplanting porcine encephalomyocarditis virus (EMCV)-infected porcine islet cells (PICs) results in transmission of the virus to recipient mice, which is manifested by acute fatal infection within 5 to 8 days. Here, we determined PIC susceptibility to a related and highly prevalent human picornavirus, coxsackie B-5 virus (CVB-5). METHODS: PICs were inoculated with CVB-5 in vitro for up to 96 hours and infectivity, level of virus replication, and cellular function determined. Subsequently, monoclonal and polyclonal antibody blocking experiments were used to investigate the receptor CVB-5 uses to enter PICs, and the ability of CVB-5-infected islets to reverse diabetes analyzed in mice. RESULTS: Adult pig islets inoculated with CVB-5 in vitro showed a typical picornaviral replication cycle with a 2-h lag phase followed by a 4-h exponential phase during which the virus titer increased by 4 logs. However, CVB-5 was less cytolytic to PICs than EMCV, resulting in a persistent productive infection lasting for up to 96 h, with minimal evidence of cell lysis. Double immunostaining confirmed the presence of CVB-5 antigens in insulin-producing islets. Infection of PICs in the presence of antibodies against human coxsackie-adenovirus receptor (CAR) resulted in near complete blockage in production of infectious virus particles whereas blocking with anti-porcine decay-accelerating factor (DAF, also called CD55) or anti-porcine membrane cofactor protein (MCP, also called CD46) only slightly decreased the number of infectious CVB-5 particles produced. Immunofluoresence staining showed CAR and MCP expression on the islet surface, but not DAF. Transplanting CVB-5-infected PICs into diabetic C57BL/6 mice resulted in reversal of diabetes. CONCLUSION: Although PICs are susceptible to human CVB-5, the infection does not appear to affect xenograft function in vitro or in vivo in the short term.