With viral infections, multiple related viral strains are often present due to coinfection or within-host evolution. We describe Haploflow, a deBruijn graph-based assembler for de novo genome assembly of viral strains from mixed sequence samples using a novel flow algorithm. We assess Haploflow across multiple benchmark data sets of increasing complexity, showing that Haploflow is faster and more accurate than viral haplotype assemblers and generic metagenome assemblers not aiming to reconstruct strains. We show Haploflow reconstructs viral strain genomes from patient HCMV samples and SARS-CoV-2 wastewater samples identical to clinical isolates.

The transmission of Macacine alphaherpesvirus 1 (McHV-1) from macaques, the natural host, to humans causes encephalitis. In contrast, human infection with Cercopithecine alphaherpesvirus 2 (CeHV-2), a closely related alphaherpesvirus from African vervet monkeys and baboons, has not been reported and it is believed that CeHV-2 is apathogenic in humans. The reasons for the differential neurovirulence of McHV-1 and CeHV-2 have not been explored on a molecular level, in part due to the absence of systems for the production of recombinant viruses. Here, we report the generation of a fosmid-based system for rescue of recombinant CeHV-2. Moreover, we show that, in this system, recombineering can be used to equip CeHV-2 with reporter genes. The recombinant CeHV-2 viruses replicated with the same efficiency as uncloned, wt virus and allowed the identification of cell lines that are highly susceptible to CeHV-2 infection. Collectively, we report a system that allows rescue and genetic modification of CeHV-2 and likely other alphaherpesviruses. This system should aid future analysis of CeHV-2 biology.

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