Certain monoclonal antibodies specific for glycoprotein D of herpes simplex virus have potent neutralizing activity but fail to block attachment of virus to cells. Here we have investigated the fate of neutralized and infectious virus after attachment to primate cells. Infectious virions fused with the cell surface such that naked nucleocapsids were detectable in the cytoplasm near or just under the plasma membrane. Neutralized virions did not fuse with the cell. They remained attached to the cell surface and could be rendered infectious by treatment with polyethylene glycol. We conclude that some anti-glycoprotein D neutralizing antibodies can inhibit the penetration of herpes simplex virus by blocking fusion of the virion envelope with the plasma membrane. These results identify a pathway of entry that initiates successful herpes simplex virus infection and a step in this pathway that is highly sensitive to neutralizing antibodies. A role for glycoprotein D in virion-cell fusion is indicated.Investigation of the mechanisms by which antibodies neutralize viral infectivity can provide information about route of entry of the virus into a cell as well as identify steps in the infectious process that can be blocked by antibodies. For enveloped viruses, entry leading to successful infection requires attachment to the cell and penetration of the nucleocapsid into the cytoplasm by fusion of the virion envelope with a cell membrane. In some instances, endocytosis of the virion may be a prerequisite for this membrane fusion, so that nucleocapsid penetration occurs by fusion of the virion envelope with the membrane of an endosome. The low pH of endosomes may be required to trigger the fusion activity (see ref. 1 for review).For other enveloped viruses, nucleocapsid penetration by fusion of the virion envelope directly with the plasma membrane has been observed by EM (2-5). It is difficult to prove, however, that such events observed by EM actually lead to viral gene expression and a replicative cycle. Here we provide evidence that fusion of herpes simplex virus (HSV) with the plasma membrane of human and monkey cells is relevant to infection. We found that fusion of HSV with the cell surface (but not virus attachment) is blocked by certain neutralizing monoclonal antibodies (mAbs).More than six membrane glycoproteins are present in the envelope of HSV, the causative agent of oral and genital herpetic lesions and, more rarely, systemic infection and encephalitis. Antibodies capable of neutralizing HSV infectivity in the absence of complement are specific for one of several glycoproteins, including gB, gD, and gH (see ref. 6 for review). Other HSV envelope glycoproteins seem not to be targets of complement-independent neutralizing antibodies.We have previously characterized eight mAbs specific for gD (Table 1). These mAbs fall into two groups-those that neutralize infectivity at relatively low concentrations without inhibiting virion adsorption to cells (first five listed) and those that neutralize and inhibit viri...

An integrated microfluidic device capable of performing a variety of genetic assays has been developed as a step towards building systems for widespread dissemination. The device integrates fluidic and thermal components such as heaters, temperature sensors, and addressable valves to control two nanoliter reactors in series followed by an electrophoretic separation. This combination of components is suitable for a variety of genetic analyses. As an example, we have successfully identified sequence-specific hemagglutinin A subtype for the A/LA/1/87 strain of influenza virus. The device uses a compact design and mass production technologies, making it an attractive platform for a variety of widely disseminated applications.

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We examined the entry process of herpes simplex virus type 1 (HSV-1) by using infectious virus and previously characterized noninfectious viruses that can bind to cells but cannot penetrate as a result of inactivation of essential viral glycoprotein D (gD) or H (gH). After contact of infectious virus with the cell plasma membrane, discernible changes of the envelope and tegument could be seen by electron microscopy. Noninfectious virions were arrested at distinct steps in interactions with cells. Viruses inactivated by anti-gD neutralizing antibodies attached to cells but were arrested prior to initiation of a visible fusion bridge between the virus and cell. As judged from its increased sensitivity to elution, virus lacking gD was less stably bound to cells than was virus containing gD. Moreover, soluble gD could substantially reduce virus attachment when added to cells prior to or with the addition of virus. Virus inactivated by anti-gH neutralizing antibodies attached and could form a fusion bridge but did not show expansion of the fusion bridge or extensive rearrangement of the envelope and tegument. We propose a model for infectious entry of HSV-1 by a series of interactions between the virion envelope and the cell plasma membrane that trigger virion disassembly, membrane fusion, and capsid penetration. In this entry process, gD mediates a stable attachment that is likely required for penetration, and gH seems to participate in fusion initiation or expansion.

Polyclonal and monoclonal antibodies to individual herpes simplex virus (HSV) glycoproteins were tested for ability to inhibit adsorption of radiolabeled HSV type 1 (HSV-1) strain HFEMsyn [HSV-1(HFEM)syn] to HEp-2 cell monolayers. Polyclonal rabbit antibodies specific for glycoprotein D (gD) or gC and three monoclonal mouse antibodies specific for gD-l or gC-1 most effectively inhibited HSV-1 adsorption. Antibodies of other specificities had less or no inhibitory activity despite demonstrable binding of the antibodies to virions. Nonimmune rabbit immunoglobulin G and Fc fragments partially inhibited adsorption when used at relatively high concentrations. These results suggest involvement of gD, gC, and perhaps gE (the Fc-binding glycoprotein) in adsorption. The monoclonal anti-gD antibodies that were most effective at inhibiting HSV-1 adsorption had only weak neutralizing activity. The most potent anti-gD neutralizing antibodies had little effect on adsorption at concentrations significantly higher than those required for neutralization. This suggests that, although some anti-gD antibodies can neutralize virus by blocking adsorption, a more important mechanism of neutralization by anti-gD antibodies may be interference with a step subsequent to adsorption, possibly penetration.