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␥-Herpesviruses, Epstein-Barr virus, and Kaposi's sarcoma-associated herpesvirus are important human pathogens, because they are involved in tumor development. Murine ␥-herpesvirus-68 (MHV-68 or ␥HV-68) has emerged as a small animal model system for the study of ␥-herpesvirus pathogenesis and host-virus interactions. To identify the genes required for viral replication in vitro and in vivo, we generated 1,152 mutants using signature-tagged transposon mutagenesis on an infectious bacterial artificial chromosome of MHV-68. Almost every ORF was mutated by random insertion. For each ORF, a mutant with an insertion proximal to the N terminus of each ORF was examined for the ability to grow in fibroblasts. Our results indicate that 41 genes are essential for in vitro growth, whereas 26 are nonessential and 6 attenuated. Replication-competent mutants were pooled to infect mice, which led to the discovery of ORF 54 being important for MHV-68 to replicate in the lung. This genetic analysis of a tumor-associated herpesvirus at the whole genome level validates signature-tagged transposon mutagenesis screening as an effective genetic system to identify important virulent genes in vivo and define interactions with the host immune system. functional mapping ͉ herpesvirus ͉ bacterial artificial chromosome ͉ deoxyuridine-triphosphatase ͉ transposition

In the foodborne pathogen , arsenic resistance is encountered primarily in serotype 4b clones considered to have enhanced virulence and is associated with an arsenic resistance gene cluster within a 35-kb chromosomal region, genomic island 2 (LGI2). LGI2 was first identified in strain Scott A and includes genes putatively involved in arsenic and cadmium resistance, DNA integration, conjugation, and pathogenicity. However, the genomic localization and sequence content of LGI2 remain poorly characterized. Here we investigated 85 arsenic-resistant strains, mostly of serotype 4b. All but one of the 70 serotype 4b strains belonged to clonal complex 1 (CC1), CC2, and CC4, three major clones associated with enhanced virulence. PCR analysis suggested that 53 strains (62.4%) harbored an island highly similar to LGI2 of Scott A, frequently (42/53) in the same location as Scott A ( homolog). Random-primed PCR and whole-genome sequencing revealed seven novel insertion sites, mostly internal to chromosomal coding sequences, among strains harboring LGI2 outside the homolog. Interestingly, many CC1 strains harbored a noticeably diversified LGI2 (LGI2-1) in a unique location ( homolog) and with a novel additional gene. With few exceptions, the tested LGI2 genes were not detected in arsenic-resistant strains of serogroup 1/2, which instead often harbored a Tn-associated arsenic resistance determinant not encountered in serotype 4b. These findings indicate that in , LGI2 has a propensity for certain serotype 4b clones, exhibits content diversity, and is highly promiscuous, suggesting an ability to mobilize various accessory genes into diverse chromosomal loci. is widely distributed in the environment and causes listeriosis, a foodborne disease with high mortality and morbidity. Arsenic and other heavy metals can powerfully shape the populations of human pathogens with pronounced environmental lifestyles such as Arsenic resistance is encountered primarily in certain serotype 4b clones considered to have enhanced virulence and is associated with a large chromosomal island, genomic island 2 (LGI2). LGI2 also harbors a cadmium resistance cassette and genes putatively involved in DNA integration, conjugation, and pathogenicity. Our findings indicate that LGI2 exhibits pronounced content plasticity and is capable of transferring various accessory genes into diverse chromosomal locations. LGI2 may serve as a paradigm on how exposure to a potent environmental toxicant such as arsenic may have dynamically selected for arsenic-resistant subpopulations in certain clones of which also contribute significantly to disease.