Rapid Hypothesis Testing in Candida albicans Clinical Isolates Using a Cloning-Free, Modular, and Recyclable System for CRISPR-Cas9 Mediated Mutant and Revertant Construction.

As increasing evidence emerges that interstrain genetic diversity among Candida albicans clinical isolates underpins phenotypic variation compared to the reference isolate SC5314, new genetic tools are required to interrogate gene function across strain backgrounds. Here, the SAT1-flipper plasmid was reengineered to contain a C. albicans codon optimized hygromycin B resistance gene (CaHygB). Cassettes were PCR-amplified from both SAT1-flipper and CaHygB-flipper plasmids using primers with homologous sequences flanking target genes of interest to serve as repair templates. Ribonucleoprotein (RNP) complexes containing proprietary CRISPR RNAs (crRNAs), universal transactivating CRISPR RNA (tracrRNA), and Cas9 protein were assembled in vitro and transformed, along with both repair templates, by electroporation into C. albicans. Homozygous deletion of the ADE2 gene results in red-pigmented colonies and this gene was used to validate our approach. Both in SC5314 and a variety of clinical isolates (529L, JS15, SJCA1, TW1), homozygous gene targeting was nearly 100% when plating on media containing nourseothricin and hygromycin B with transformation efficiencies exceeding 104 homozygous deletion mutants per µg of DNA. A gene reversion system was also employed with plasmids pDUP3 and pDIS3 engineered to contain the ADH1 terminator and an overlap extension PCR-mediated approach combined with CRISPR-Cas9 targeting at the NEUT5 neutral locus. A variety of single or compound mutants (Δ/Δals3, Δ/Δcph1 Δ/Δefg1, Δ/Δece1) and their revertant strains were constructed and phenotypically validated by a variety of assays, including biofilm formation, hyphal growth, and macrophage IL-1ß response. Thus, we have established a cloning-free, modular system for highly efficient homozygous gene deletion and reversion in diverse isolates. IMPORTANCE Recently, phenotypic heterogeneity in Candida albicans isolates has been recognized as an underappreciated factor contributing to gene diversification and broadly impacts strain-to-strain antifungal resistance, fitness, and pathogenicity. We have designed a cloning-free genetic system for rapid gene deletion and reversion in C. albicans clinical isolates that interlaces established recyclable genetic systems with CRISPR-Cas9 technology. The SAT1-flipper was reengineered to contain CaHygB encoding resistance to hygromycin B. Using a modular PCR-mediated approach coupled with in vitro ribonucleoprotein assembly with commercial reagents, both SAT1- and CaHygB-flipper cassettes were simultaneously integrated at loci with high efficiency (104 transformants per µg DNA) and upward of 99% homozygous gene targeting across a collection of diverse isolates of various anatomical origin. Revertant strains were constructed by overlap extension PCR with CRISPR-Cas9 targeted integration at the NEUT5 locus. Thus, this facile system will aid in unraveling the genetic factors contributing to the complexity of intraspecies diversity.

Transplacental congenital human herpesvirus 6 infection caused by maternal chromosomally integrated virus.

Congenital human herpesvirus 6 (HHV-6) infection results from germline passage of chromosomally integrated HHV-6 (CI-HHV-6) and from transplacental passage of maternal HHV-6 infection. We aimed to determine whether CI-HHV-6 could replicate and cause transplacentally acquired HHV-6 infection. HHV-6 DNA, variant type, and viral loads were determined with samples (cord blood, peripheral blood, saliva, urine, and hair) obtained from 6 infants with transplacentally acquired HHV-6 and with samples of their parents' hair. No fathers but all mothers of infants with transplacentally acquired HHV-6 had CI-HHV-6, and the mother's CI-HHV-6 variant was the same variant causing the transplacentally acquired congenital HHV-6 infection. This suggests the possibility that CI-HHV-6 replicates and may cause most, if not all, congenital HHV-6 infections.

Chromosomal integration of human herpesvirus 6 is the major mode of congenital human herpesvirus 6 infection.

OBJECTIVE: We examined the frequency and characteristics of chromosomally integrated human herpesvirus 6 among congenitally infected children. METHODS: Infants with and without congenital human herpesvirus 6 infection were prospectively monitored. Cord blood mononuclear cell, peripheral blood mononuclear cell, saliva, urine, and hair follicle samples were examined for human herpesvirus 6 DNA. Human herpesvirus 6 RNA, serum antibody, and chromosomally integrated human herpesvirus 6 levels were also assessed. RESULTS: Among 85 infants, 43 had congenital infections and 42 had postnatal infections. Most congenital infections (86%) resulted from chromosomally integrated human herpesvirus 6; 6 infants (14%) had transplacental infections. Children with chromosomally integrated human herpesvirus 6 had high viral loads in all sites (mean: 5-6 log(10) genomic copies per mug of cellular DNA); among children with transplacental infection or postnatal infection, human herpesvirus 6 DNA was absent in hair samples and inconsistent in other samples, and viral loads were significantly lower. One parent of each child with chromosomally integrated human herpesvirus 6 who had parental hair samples tested had hair containing human herpesvirus 6 DNA. Variant A caused 32% of chromosomally integrated human herpesvirus 6 infections, compared with 2% of postnatal infections. Replicating human herpesvirus 6 was detected only among chromosomally integrated human herpesvirus 6 samples (8% of cord blood mononuclear cells and peripheral blood mononuclear cells). Cord blood human herpesvirus 6 antibody levels were similar among children with chromosomally integrated human herpesvirus 6, transplacental infection, and postnatal infection and between children with maternal and paternal chromosomally integrated human herpesvirus 6 transmission. CONCLUSIONS: Human herpesvirus 6 congenital infection results primarily from chromosomally integrated virus which is passed through the germ-line. Infants with chromosomally integrated human herpesvirus 6 had high viral loads in all specimens, produced human herpesvirus 6 antibody, and mRNA. The clinical relevance needs study as 1 of 116 newborns may have chromosomally integrated human herpesvirus 6 blood specimens.

Characteristics and acquisition of human herpesvirus (HHV) 7 infections in relation to infection with HHV-6.

Although both human herpesvirus (HHV) 6 and HHV-7 infections are ubiquitous during childhood, few acute HHV-7 infections are identified. It is unknown whether HHV-7 viremia indicates primary infection, as with HHV-6, or reactivation, and if these differ clinically. We studied, in otherwise healthy children < or =10 years old, HHV-7 and HHV-6 infections and their interaction by serologic assessment, viral isolation, and polymerase chain reaction. In children < or =24 months of age, HHV-7 infections occurred less often than HHV-6 infections (P< or =.002). Of 2806 samples from 2365 children < or =10 years old, 30 (1%) showed evidence of HHV-7 viremia; 23 (77%) of these were primary and 7 (23%) were reactivated HHV-7 infections. Four (13%) showed concurrent HHV-6 viremia, 2 associated with primary HHV-7 infections. The clinical manifestations of primary and reactivated HHV-7 infections were similar, except that seizures occurred more frequently in reactivated infections. These findings, previously unrecognized in otherwise healthy children, suggest that HHV-7 viremia could represent primary or reactivated infection and may be affected by the interaction between HHV-6 and HHV-7.

Congenital infections with human herpesvirus 6 (HHV6) and human herpesvirus 7 (HHV7).

OBJECTIVE: To examine whether: (1) congenital human herpesvirus 6 (HHV6) and human herpesvirus 7 (HHV7) infections occur; whether (2) their manifestations differ from postnatal infections; and whether (3) HHV6 and HHV7 infections differ despite their close relatedness. STUDY DESIGN: HHV6 and HHV7 infections acquired congenitally and postnatally in normal children were compared using viral isolation, serology, reverse-transcription polymerase chain reaction (RT-PCR) and nested DNA-PCR for HHV6 variant A (HHV6A), HHV6 variant B (HHV6B), and HHV7. RESULTS: HHV6 DNA was detected in 57 (1%) of 5638 cord bloods. HHV7 DNA, however, was not detected in 2129 cord bloods. Congenital HHV6 infections differed from postnatal infections, which were acute febrile illnesses. Congenital infections were asymptomatic, 10% demonstrated reactivation at birth, and HHV6 DNA persistence in follow-up blood samples was significantly more frequent. One-third of congenital infections were HHV6A, whereas all postnatal infections were HHV6B. CONCLUSIONS: Congenital HHV6 infections occurred in 1% of births, similar to the rate for cytomegalovirus infection. Congenital infections were clinically and virologically distinct from postnatal infections. Congenital HHV7 infections, however, were not detected, suggesting considerable differences in transmission and pathogenesis in these closely related beta-herpesviruses.

Human herpesvirus 6 (HHV6) DNA persistence and reactivation in healthy children.

OBJECTIVE: To determine in healthy children after primary infection the persistence of human herpesvirus 6 (HHV6) DNA, the presence and frequency of HHV6 re-activation or re-infection, and the relationship of both to illness and the presence of human herpesvirus 7 (HHV7) infection. STUDY DESIGN: Children 1 to 12 years of age with previous HHV6 infection were prospectively evaluated by HHV6 and HHV7 DNA polymerase chain reaction (PCR) and reverse transcription (RT)-PCR for HHV6. HHV6 plasma antibody titers were measured. Illnesses were recorded by diary, and physician records were reviewed. RESULTS: HHV6 DNA was detected in >or=1 peripheral blood mononuclear cell (PBMC) samples in 89% of children. HHV6 reactivation and re-infection were detected by RT-PCR in 1.1% of samples. Detection of HHV6 DNA was intermittent in 76% of children and was not associated with cumulative rates of illness. Illness at a study visit was significantly associated with the absence of HHV6 and HHV7 DNA in PBMC samples and was not associated with HHV6 antibody titer or the presence of HHV6 DNA in saliva. CONCLUSIONS: HHV6 DNA persists in most children intermittently following primary infection and is unrelated to illness. Reactivation of HHV6 occurs in healthy children without apparent illness.