Embryonic stem cell (ESC) pluripotency is dependent on an intrinsic gene regulatory network centered on Oct4. Propagation of the pluripotent state is stimulated by the cytokine leukemia inhibitory factor (LIF) acting through the transcriptional regulator Stat3. Here, we show that this extrinsic stimulus converges with the intrinsic circuitry in Krüppel-factor activation. Oct4 primarily induces Klf2 while LIF/Stat3 selectively enhances Klf4 expression. Overexpression of either factor reduces LIF dependence, but with quantitative and qualitative differences. Unlike Klf4, Klf2 increases ESC clonogenicity, maintains undifferentiated ESCs in the genetic absence of Stat3, and confers resistance to BMP-induced differentiation. ESCs expanded with Klf2 remain capable of contributing to adult chimeras. Postimplantation-embryo-derived EpiSCs lack both Klf2 and Klf4 and expression of either can reinstate naive pluripotency. These findings indicate that Oct4 and Stat3 intersect in directing expression of Klf transcriptional regulators with overlapping properties that additively reinforce ground-state ESC pluripotency, identity, and self-renewal.

Correspondence n engl j med 375;5 nejm.org August 4, 2016 Human Coronavirus OC43 Associated with Fatal EncephalitisTo the Editor: Encephalitis is a serious neurologic syndrome characterized by brain inflammation that may be fatal. Although the majority of cases are caused by viruses, the identification of a causal organism can be difficult. Encephalopathy that affects patients with immunodeficiency is particularly challenging to diagnose, since the clinical presentation may be atypical and the differential diagnosis may include unusual pathogens or a noninfective cause. Deep sequencing of clinical samples has the potential to identify the pathogens associated with encephalitis, particularly in cases in which traditional techniques have not identified the candidate causative pathogen. 1Here we report the use of deep sequencing of a brain biopsy sample obtained from an 11-monthold boy with severe combined immunodeficiency who had symptoms of viral encephalitis with negative results on conventional diagnostic polymerase-chain-reaction (PCR) assay. The boy's family provided written informed consent.The boy underwent unconditioned cord-blood transplantation, which resulted in T-cell engraftment. Nonetheless, his condition continued to deteriorate, and he died 1.5 months after receiving the transplant. RNA sequencing of a brain biopsy sample obtained 2 months after the onset of symptoms showed the presence of human coronavirus OC43 (HCoV-OC43), which was sub- sequently confirmed on real-time PCR (threshold cycle, 24) and brain immunohistochemical analysis (Fig. 1). Full details on the case history, library preparation, bioinformatics analysis, species identification as reported by the profiling method metaMix, 2 PCR confirmation, immunohistochemical analysis, and phylogenetic and variant analyses are provided in the Supplementary Appendix, available with the full text of this letter at NEJM.org.The human betacoronaviruses, including HCoV-OC43, are predominantly associated with respiratory tract infections. The group includes viruses that cause the severe acute respiratory syndrome and the Middle East respiratory syndrome. HCoV-OC43 is generally associated with mild upper respiratory tract infections, although it has been shown to have neuroinvasive properties. In vivo studies in mice have shown that HCoV-OC43 can infect neurons and cause encephalitis.3 The virus has also been shown to cause persistent infections in human neural-cell lines. 4 A single report identified HCoV-OC43 RNA in the cerebrospinal fluid of a child with acute disseminated encephalomyelitis. 5 In the case we describe here, three independent methods were used to identify HCoV-OC43 in brain tissue of a child with acute encephalomyelitis.Deep sequencing of biopsy material provides an important tool for the diagnosis of unexplained encephalomyelitis, particularly in patients with immunodeficiency who have undergone stem-cell transplantation, when the differential diagnosis may include immune-mediated inflammation or drug toxicity. The identificatio...

Type I interferon (IFN-α/β) is a fundamental antiviral defense mechanism. Mouse models have been pivotal to understanding the role of IFN-α/β in immunity, although validation of these findings in humans has been limited. We investigated a previously healthy child with fatal encephalitis following inoculation of the live-attenuated measles, mumps and rubella (MMR) vaccine. By targeted resequencing we identified a homozygous mutation in the high-affinity * Correspondence to: christopher.duncan@ncl.ac.uk or sophie.hambleton@ncl.ac.uk.

Brain biopsy from a child with unknown cause of encephalopathy was deep-sequenced. Astrovirus VA1/HMO-C was identified, highly divergent from human astroviruses and 95% identical to astrovirus previously associated with encephalitis. Findings suggest astrovirus VA1/HMO-C is an under-recognized cause of viral encephalitis.