Zika virus (ZIKV), a mosquito-transmitted flavivirus responsible for sporadic outbreaks of mild and febrile illness in Africa and Asia, reemerged in the last decade causing serious human diseases, including microcephaly, congenital malformations, and Guillain-Barré syndrome. Although genomic and phylogenetic analyses suggest that genetic evolution may have led to the enhanced virulence of ZIKV, experimental evidence supporting the role of specific genetic changes in virulence is currently lacking. One sequence motif, VNDT, containing an N-linked glycosylation site in the envelope (E) protein, is polymorphic; it is absent in many of the African isolates but present in all isolates from the recent outbreaks. In the present study, we investigated the roles of this sequence motif and glycosylation of the E protein in the pathogenicity of ZIKV. We first constructed a stable full-length cDNA clone of ZIKV in a novel linear vector from which infectious virus was recovered. The recombinant ZIKV generated from the infectious clone, which contains the VNDT motif, is highly pathogenic and causes lethality in a mouse model. In contrast, recombinant viruses from which the VNDT motif is deleted or in which the N-linked glycosylation site is mutated by single-amino-acid substitution are highly attenuated and nonlethal. The mutant viruses replicate poorly in the brains of infected mice when inoculated subcutaneously but replicate well following intracranial inoculation. Our findings provide the first evidence that N-linked glycosylation of the E protein is an important determinant of ZIKV virulence and neuroinvasion.IMPORTANCE The recent emergence of Zika virus (ZIKV) in the Americas has caused major worldwide public health concern. The virus appears to have gained significant pathogenicity, causing serious human diseases, including microcephaly and Guillain-Barré syndrome. The factors responsible for the emergence of pathogenic ZIKV are not understood at this time, although genetic changes have been shown to facilitate virus transmission. All isolates from the recent outbreaks contain an N-linked glycosylation site within the viral envelope (E) protein, whereas many isolates of the African lineage virus lack this site. To elucidate the functional significance of glycosylation in ZIKV pathogenicity, recombinant ZIKVs from infectious clones with or without the glycan on the E protein were generated. ZIKVs lacking the glycan were highly attenuated for the ability to cause mortality in a mouse model and were severely compromised for neuroinvasion. Our studies suggest glycosylation of the E protein is an important factor contributing to ZIKV pathogenicity.

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myocarditis, affected individuals can develop dilated cardiomyopathy (DCM) [4]. Approximately half of patients with DCM undergo heart transplantations due to the lack of effective chemotherapy. Enteroviruses are commonly suspected in DCM patients because the genomic material can be detected in up to 70% of patients, and serologically, virus-reactive neutralizing antibodies can be found in 50% of patients [2]. The question to be addressed is how virus infection can promote DCM. Auto immunity is one possible mechanism, as evidenced in patients with myocarditis/DCM by the presence of autoantibodies to various cardiac antigens, such as cardiac myosin heavy chain (Myhc)-α, adenine nucleotide translocator (ANT), β-adrenergic receptor-1, branched chain α-ketoacid dehydrogenase (BCKD), laminin, and muscarinic receptor; Myhc-α is a well-characterized antigen [5,6]. Based on cellular infiltrations, forms of myocarditis have been classified as lymphocytic, giant cell, and eosinophilic, and various infectious (viruses, bacteria, protozoa, helminthes) and noninfectious (drugs, metals, chemicals) agents

Epitope from Acanthamoeba castellanii That Cross-React with Proteolipid Protein 139-151-Reactive T Cells Induces Autoimmune Encephalomyelitis in SJL Mice" (2010). Jay Reddy Publications. 17. https://digitalcommons.unl.edu/vbsjayreddy/17 tein databases. This search resulted in the identification of one novel peptide spanning aa 83-95 of rhodanese-related sulfurtransferase in Acanthamoeba castellanii (ACA) and it induces EAE similar to that of PLP 139-151. Materials and methods MiceFour to six-week-old female SJL/J (H-2 s ) mice were obtained from the Jackson Laboratory (Bar Harbor, Maine). The mice were maintained in accordance with the animal protocol guidelines of the University of Nebraska-Lincoln, Lincoln, Nebraska. Peptide synthesis and immunization proceduresPLP 139-151 (HSLGKWLGHPDKF), ACA 83-95 (YFLLKWLGH-PNVS) and neuraminidase (NASE) 101-120 (EALVRQGLAKVAY-VYKPNNT) were synthesized on 9-fluorenylmethyloxycarbonyl chemistry (Neopeptide, Cambridge, Massachusetts). All peptides were HPLC-purified (N90%) and confirmed by mass spectroscopy. To measure recall responses 100 µg of each peptide emulsified in CFA was administered subcutaneously in the flank. For disease induction, Mycobacterium tuberculosis (MTB) H37RA extract (Difco Laboratories, Detroit, Michigan) was added as an additional component to a final concentration of 5 mg/ml. Pertussis toxin (List Biological Laboratories, Campbell, California) was administered (100 ng per mouse) intraperitoneally on day 0 and day 2 postimmunization. Identification of microbial peptides that mimic PLP 139-151PLP 139-151 is an immunodominant epitope in which the critical residues required for major histocompatibility complex (MHC) class II and T cell receptor (TCR)-binding have been well-characterized (Figure 1). By using PLP 139-151 as a putative antigen, we performed pattern search using the prosite scan of the Bioinformatics Toolkit

BackgroundOsteopetrosis is a skeletal disorder of humans and animals characterized by the formation of overly dense bones, resulting from a deficiency in the number and/or function of bone-resorbing osteoclast cells. In cattle, osteopetrosis can either be induced during gestation by viral infection of the dam, or inherited as a recessive defect. Genetically affected calves are typically aborted late in gestation, display skull deformities and exhibit a marked reduction of osteoclasts. Although mutations in several genes are associated with osteopetrosis in humans and mice, the genetic basis of the cattle disorder was previously unknown.ResultsWe have conducted a whole-genome association analysis to identify the mutation responsible for inherited osteopetrosis in Red Angus cattle. Analysis of >54,000 SNP genotypes for each of seven affected calves and nine control animals localized the defective gene to the telomeric end of bovine chromosome 4 (BTA4). Homozygosity analysis refined the interval to a 3.4-Mb region containing the SLC4A2 gene, encoding an anion exchanger protein necessary for proper osteoclast function. Examination of SLC4A2 from normal and affected animals revealed a ~2.8-kb deletion mutation in affected calves that encompasses exon 2 and nearly half of exon 3, predicted to prevent normal protein function. Analysis of RNA from a proven heterozygous individual confirmed the presence of transcripts lacking exons 2 and 3, in addition to normal transcripts. Genotyping of additional animals demonstrated complete concordance of the homozygous deletion genotype with the osteopetrosis phenotype. Histological examination of affected tissues revealed scarce, morphologically abnormal osteoclasts displaying evidence of apoptosis.ConclusionsThese results indicate that a deletion mutation within bovine SLC4A2 is associated with osteopetrosis in Red Angus cattle. Loss of SLC4A2 function appears to induce premature cell death, and likely results in cytoplasmic alkalinization of osteoclasts which, in turn, may disrupt acidification of resorption lacunae.

Among equids with WNV infection, age, vaccination status, an inability to rise, and sex were associated with the risk of death.

Heart failure, a leading cause of death in humans, can emanate from myocarditis. Although most individuals with myocarditis recover spontaneously, some develop chronic dilated cardiomyopathy. Myocarditis may result from both infectious and noninfectious causes, including autoimmune responses to cardiac antigens. In support of this notion, intracellular cardiac antigens, like cardiac myosin heavy chain-a, cardiac troponin-I, and adenine nucleotide translocator 1 (ANT 1 ), have been identified as autoantigens in cardiac autoimmunity. Herein, we demonstrate that ANT 1 can induce autoimmune myocarditis in A/J mice by generating autoreactive T cells. We show that ANT 1 encompasses multiple immunodominant epitopes (namely, ANT 1 21-40, ANT 1 31-50, ANT 1 171-190, and ANT 1 181-200). Although all four peptides induce comparable T-cell responses, only ANT 1 21-40 was found to be a major myocarditogenic epitope in immunized animals. The myocarditis-inducing ability of ANT 1 21-40 was associated with the generation of T cells producing predominantly IL-17A, and the antigen-sensitized T cells could transfer the disease to naïve recipients. These data indicate that cardiac mitochondrial proteins can be target autoantigens in myocarditis, supporting the notion that the antigens released as a result of primary damage may contribute to the persistence of chronic inflammation through autoimmunity. Myocarditis can occur as a result of exposure to various infectious and noninfectious insults, but does not generally lead to a fatal outcome (ie, most affected individuals can recover spontaneously). However, a proportion of those affected can develop dilated cardiomyopathy (DCM). Estimates indicate that approximately half of DCM patients undergo heart transplantation because of a lack of alternative therapeutic options.1e3 Furthermore, several clinical studies suggest that DCM patients can have autoantibodies to several cardiac antigens, including adenine nucleotide translocator (ANT).4e6 Because DCM can arise as a sequel to myocarditis, it has been postulated that autoimmune response may be an underlying mechanism in its pathogenesis. 7ANT exists in multiple isoforms, all four of which are expressed in humans (ANT 1 , ANT 2 , ANT 3 , and ANT 4 ), but only three in mice (ANT 1 , ANT 2 , and ANT 4 ). ANT 1 is expressed in muscle tissues (heart and skeletal) and the brain, ANT 2 can be expressed in liver, kidney, and heart, and ANT 4 expression is restricted to the testes in mice.

Mulefoot disease (MFD) is an autosomal recessive disorder of phenotypically variable expression that causes syndactyly in certain strains of cows. MFD maps to a narrow interval on bovine chromosome 15 that is syntenic to human chromosome 11p12-p11.2. This region contains MEGF7/LRP4 (approved gene symbol LRP4), a gene that encodes a member of the multifunctional low-density lipoprotein receptor gene family. Targeted and naturally occurring mutations in the murine Megf7/Lrp4 gene, a putative coreceptor in the Wnt signaling pathway, cause polysyndactyly in the rodent. Thus, Megf7/Lrp4 is a strong candidate for the MFD mutation. Using PCR analysis of tissue samples and sperm from confirmed homozygous MFD carriers, we have identified a functional single base pair mutation in the affected animals. We show that a G --> A transition at the first nucleotide in the splice donor site of intron 37 completely disables this splice site. The abnormal splicing that is caused by this mutation predicts the generation of a dysfunctional membrane-anchored receptor lacking the normal cytoplasmic domain. These findings confirm that autosomal recessive loss-of-function mutations in Megf7/Lrp4 result in phenotypically similar forms of syndactyly in different mammalian species and that such mutations are the cause of MFD in bovines.