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To detect human herpesvirus (HHV)-8/Kaposi's sarcoma-associated herpesvirus (KSHV) neutralizing antibodies (nAb).Antibodies capable of inhibiting HHV-8 infection were measured by infecting transformed dermal microvascular endothelial cells (tDMVEC) with HHV-8 that had been pre-incubated with serum from HHV-8-seropositive or -seronegative subjects. The level of infection was quantified 48 h later. HHV-8 was prepared from JSC-1 primary effusion lymphoma cells; the titre of enveloped virions was determined by electron microscopy. Virus was incubated with serum samples for 60 min before inoculating tDMVEC. The level of infection was quantified by indirect immunofluorescence assay, staining for HHV-8 latency-associated nuclear antigen (LANA)-1. Inhibition of infection was determined by comparing the level of infection obtained with HHV-8-seropositive subject serum with the level obtained by incubation with seronegative serum. Up to 61% of cells were infected with HHV-8 in the absence of human serum; this level was not affected by pre-incubating the virus with HHV-8-seronegative serum. At dilutions of 1 : 10 and 1 : 50, HHV-8-seropositive sera significantly inhibited infection compared to seronegative controls (P = 0.036 for both serum dilutions, Mann–Whitney). The endpoint of inhibition was 1 : 100, when the serum of one of five subjects inhibited virus infection. At 1 : 500 dilution, there was no difference in the level of infection after virus incubation with seropositive or seronegative sera (P = 0.578). Depletion of antibody from serum with protein A reversed the inhibitory effect, confirming it was antibody-mediated.This study is the first to identify HHV-8 antibodies in infected subjects that reduce in vitro infectivity of the virus.