Mono is spread by person-to-person contact. Saliva is the primary method of transmitting mono, which leads to the infection of B lymphocytes in the mouth and throat. Infectious mononucleosis developed its common name of "kissing disease" from this prevalent form of transmission among teenagers. It typically takes between four to eight weeks for symptoms of mono to appear after the initial infection with EBV. A person with mono can also pass the disease by coughing or sneezing, causing small droplets of infected saliva and/or mucus to be suspended in the air which can be inhaled by others. Sharing food or beverages from the same container or utensil can also transfer the virus from one person to another since contact with infected saliva may result.

Most people have been exposed to the virus as children, and as a result of the exposure, they have developed immunity to the virus. It is of note that most people who are exposed to the EBV don't ever develop mononucleosis. The incubation period for mono, meaning the time from the initial viral infection until the appearance of symptoms, is between four and eight weeks. During an infection, a person is likely able to transmit the virus to others for at least a few weeks and possibly longer, even after symptoms have disappeared (see below).

Research has shown that, depending on the method used to detect the virus, anywhere from 20%-80% of people who have had mononucleosis and have recovered will continue to secrete the EBV in their saliva for years due to periodic "reactivations" of the viral infection. Since healthy people without symptoms also secrete the virus during reactivation episodes throughout their lifetime, isolation of people infected with EBV is not necessary. It is currently believed that these healthy people, who nevertheless secrete EBV particles, are the primary reservoir for transmission of EBV among humans.

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