Vaccine Mediated Protection Against Zika Virus-Induced Congenital Disease.

The emergence of Zika virus (ZIKV) and its association with congenital malformations has prompted the rapid development of vaccines. Although efficacy with multiple viral vaccine platforms has been established in animals, no study has addressed protection during pregnancy. We tested in mice two vaccine platforms, a lipid nanoparticle-encapsulated modified mRNA vaccine encoding ZIKV prM and E genes and a live-attenuated ZIKV strain encoding an NS1 protein without glycosylation, for their ability to protect against transmission to the fetus. Vaccinated dams challenged with a heterologous ZIKV strain at embryo day 6 (E6) and evaluated at E13 showed markedly diminished levels of viral RNA in maternal, placental, and fetal tissues, which resulted in protection against placental damage and fetal demise. As modified mRNA and live-attenuated vaccine platforms can restrict in utero transmission of ZIKV in mice, their further development in humans to prevent congenital ZIKV syndrome is warranted.

A protective Zika virus E-dimer-based subunit vaccine engineered to abrogate antibody-dependent enhancement of dengue infection.

Infections with dengue virus (DENV) and Zika virus (ZIKV) can induce cross-reactive antibody responses. Two immunodominant epitopes-one to precursor membrane protein and one to the fusion loop epitope on envelope (E) protein-are recognized by cross-reactive antibodies1-3 that are not only poorly neutralizing, but can also promote increased viral replication and disease severity via Fcγ receptor-mediated infection of myeloid cells-a process termed antibody-dependent enhancement (ADE)1,4,5. ADE is a significant concern for both ZIKV and DENV vaccines as the induction of poorly neutralizing cross-reactive antibodies may prime an individual for ADE on natural infection. In this report, we describe the design and production of covalently stabilized ZIKV E dimers, which lack precursor membrane protein and do not expose the immunodominant fusion loop epitope. Immunization of mice with ZIKV E dimers induces dimer-specific antibodies, which protect against ZIKV challenge during pregnancy. Importantly, the ZIKV E-dimer-induced response does not cross-react with DENV or induce ADE of DENV infection.

Therapeutic efficacy of favipiravir against Bourbon virus in mice.

Bourbon virus (BRBV) is an emerging tick-borne RNA virus in the orthomyxoviridae family that was discovered in 2014. Although fatal human cases of BRBV have been described, little is known about its pathogenesis, and no antiviral therapies or vaccines exist. We obtained serum from a fatal case in 2017 and successfully recovered the second human infectious isolate of BRBV. Next-generation sequencing of the St. Louis isolate of BRBV (BRBV-STL) showed >99% nucleotide identity to the original reference isolate. Using BRBV-STL, we developed a small animal model to study BRBV-STL tropism in vivo and evaluated the prophylactic and therapeutic efficacy of the experimental antiviral drug favipiravir against BRBV-induced disease. Infection of Ifnar1-/- mice lacking the type I interferon receptor, but not congenic wild-type animals, resulted in uniformly fatal disease 6 to 10 days after infection. RNA in situ hybridization and viral yield assays demonstrated a broad tropism of BRBV-STL with highest levels detected in liver and spleen. In vitro replication and polymerase activity of BRBV-STL were inhibited by favipiravir. Moreover, administration of favipiravir as a prophylaxis or as post-exposure therapy three days after infection prevented BRBV-STL-induced mortality in immunocompromised Ifnar1-/- mice. These results suggest that favipiravir may be a candidate treatment for humans who become infected with BRBV.

Zika Virus Causes Acute and Chronic Prostatitis in Mice and Macaques.

BACKGROUND: Sexual transmission and persistence of Zika virus (ZIKV) in the male reproductive tract has raised concerned for potential damaging effects on function. Animal studies have demonstrated that ZIKV virus can infect and damage the testis and epididymis, and these results has been correlated to lower sperm counts in ZIKV-infected humans. The prostate plays a vital role in the male reproductive tract, with acute and chronic prostatitis linked to male infertility. METHODS: In this study, we evaluated the effects of ZIKV virus on the prostate in mice and nonhuman primates. RESULTS: In mice, ZIKV infected the prostate and triggered inflammation that persisted even after virus clearance. Evidence of chronic prostatitis associated with ZIKV infection remained for several months. Similar histological findings were observed in the prostate of ZIKV-infected rhesus macaques. CONCLUSIONS: These studies establish that ZIKV replicates in the prostate and can cause acute and chronic inflammatory and proliferative changes in mouse and nonhuman primate models.

Mutation of Influenza A Virus PA-X Decreases Pathogenicity in Chicken Embryos and Can Increase the Yield of Reassortant Candidate Vaccine Viruses.

The PA-X protein of influenza A virus has roles in host cell shutoff and viral pathogenesis. While most strains are predicted to encode PA-X, strain-dependent variations in activity have been noted. We found that PA-X protein from the A/PR/8/34 (PR8) strain had significantly lower repressive activity against cellular gene expression than PA-X proteins from the avian strains A/turkey/England/50-92/91 (H5N1) (T/E) and A/chicken/Rostock/34 (H7N1). Loss of normal PA-X expression, either by mutation of the frameshift site or by truncating the X open reading frame (ORF), had little effect on the infectious virus titer of PR8 or PR8 7:1 reassortants with T/E segment 3 grown in embryonated hens' eggs. However, in both virus backgrounds, mutation of PA-X led to decreased embryo mortality and lower overall pathology, effects that were more pronounced in the PR8 strain than in the T/E reassortant, despite the low shutoff activity of the PR8 PA-X. Purified PA-X mutant virus particles displayed an increased ratio of hemagglutinin (HA) to nucleoprotein (NP) and M1 compared to values for their wild-type (WT) counterparts, suggesting altered virion composition. When the PA-X gene was mutated in the background of poorly growing PR8 6:2 vaccine reassortant analogues containing the HA and neuraminidase (NA) segments from H1N1 2009 pandemic viruses or from an avian H7N3 strain, HA yield increased up to 2-fold. This suggests that the PR8 PA-X protein may harbor a function unrelated to host cell shutoff and that disruption of the PA-X gene has the potential to improve the HA yield of vaccine viruses.IMPORTANCE Influenza A virus is a widespread pathogen that affects both humans and a variety of animal species, causing regular epidemics and sporadic pandemics, with major public health and economic consequences. A better understanding of virus biology is therefore important. The primary control measure is vaccination, which for humans mostly relies on antigens produced in eggs from PR8-based viruses bearing the glycoprotein genes of interest. However, not all reassortants replicate well enough to supply sufficient virus antigen for demand. The significance of our research lies in identifying that mutation of the PA-X gene in the PR8 strain of virus can improve antigen yield, potentially by decreasing the pathogenicity of the virus in embryonated eggs.

Protective Efficacy of Nucleic Acid Vaccines Against Transmission of Zika Virus During Pregnancy in Mice.

Zika virus (ZIKV) caused an epidemic of congenital malformations in 2015-2016. Although many vaccine candidates have been generated, few have demonstrated efficacy against congenital ZIKV infection. Here, we evaluated lipid-encapsulated messenger RNA (mRNA) vaccines and a DNA plasmid vaccine encoding the prM-E genes of ZIKV in mouse models of congenital infection. Although the DNA vaccine provided comparable efficacy against vertical transmission of ZIKV, the mRNA vaccines, including one that minimizes antibody-dependent enhancement of infection, elicited higher levels of antigen-specific long-lived plasma cells and memory B cells. Despite the induction of robust neutralizing antibody titers by all vaccines, breakthrough seeding of the placenta and fetal head was observed in a small subset of type I interferon signaling-deficient immunocompromised dams. In comparison, evaluation of one of the mRNA vaccines in a human STAT2-knockin transgenic immunocompetent mouse showed complete protection against congenital ZIKV transmission. These data will inform ongoing human ZIKV vaccine development efforts and enhance our understanding of the correlates of vaccine-induced protection.

Heartland virus infection in a heart transplant recipient from the Heartland.

Tick-borne infections represent a significant health risk each year in the United States. Immunocompromised patients are typically at risk of more severe disease manifestations than their immunocompetent counterparts. Here we report a case of a newly emerging phlebovirus, Heartland virus, in a heart transplant recipient.

Role of the B Allele of Influenza A Virus Segment 8 in Setting Mammalian Host Range and Pathogenicity.

UNLABELLED: Two alleles of segment 8 (NS) circulate in nonchiropteran influenza A viruses. The A allele is found in avian and mammalian viruses, but the B allele is viewed as being almost exclusively found in avian viruses. This might reflect the fact that one or both of its encoded proteins (NS1 and NEP) are maladapted for replication in mammalian hosts. To test this, a number of clade A and B avian virus-derived NS segments were introduced into human H1N1 and H3N2 viruses. In no case was the peak virus titer substantially reduced following infection of various mammalian cell types. Exemplar reassortant viruses also replicated to similar titers in mice, although mice infected with viruses with the avian virus-derived segment 8s had reduced weight loss compared to that achieved in mice infected with the A/Puerto Rico/8/1934 (H1N1) parent. In vitro, the viruses coped similarly with type I interferons. Temporal proteomics analysis of cellular responses to infection showed that the avian virus-derived NS segments provoked lower levels of expression of interferon-stimulated genes in cells than wild type-derived NS segments. Thus, neither the A nor the B allele of avian virus-derived NS segments necessarily attenuates virus replication in a mammalian host, although the alleles can attenuate disease. Phylogenetic analyses identified 32 independent incursions of an avian virus-derived A allele into mammals, whereas 6 introductions of a B allele were identified. However, A-allele isolates from birds outnumbered B-allele isolates, and the relative rates of Aves-to-Mammalia transmission were not significantly different. We conclude that while the introduction of an avian virus segment 8 into mammals is a relatively rare event, the dogma of the B allele being especially restricted is misleading, with implications in the assessment of the pandemic potential of avian influenza viruses. IMPORTANCE: Influenza A virus (IAV) can adapt to poultry and mammalian species, inflicting a great socioeconomic burden on farming and health care sectors. Host adaptation likely involves multiple viral factors. Here, we investigated the role of IAV segment 8. Segment 8 has evolved into two distinct clades: the A and B alleles. The B-allele genes have previously been suggested to be restricted to avian virus species. We introduced a selection of avian virus A- and B-allele segment 8s into human H1N1 and H3N2 virus backgrounds and found that these reassortant viruses were fully competent in mammalian host systems. We also analyzed the currently available public data on the segment 8 gene distribution and found surprisingly little evidence for specific avian host restriction of the B-clade segment. We conclude that B-allele segment 8 genes are, in fact, capable of supporting infection in mammals and that they should be considered during the assessment of the pandemic risk of zoonotic influenza A viruses.

Identification of a novel splice variant form of the influenza A virus M2 ion channel with an antigenically distinct ectodomain.

Segment 7 of influenza A virus produces up to four mRNAs. Unspliced transcripts encode M1, spliced mRNA2 encodes the M2 ion channel, while protein products from spliced mRNAs 3 and 4 have not previously been identified. The M2 protein plays important roles in virus entry and assembly, and is a target for antiviral drugs and vaccination. Surprisingly, M2 is not essential for virus replication in a laboratory setting, although its loss attenuates the virus. To better understand how IAV might replicate without M2, we studied the reversion mechanism of an M2-null virus. Serial passage of a virus lacking the mRNA2 splice donor site identified a single nucleotide pseudoreverting mutation, which restored growth in cell culture and virulence in mice by upregulating mRNA4 synthesis rather than by reinstating mRNA2 production. We show that mRNA4 encodes a novel M2-related protein (designated M42) with an antigenically distinct ectodomain that can functionally replace M2 despite showing clear differences in intracellular localisation, being largely retained in the Golgi compartment. We also show that the expression of two distinct ion channel proteins is not unique to laboratory-adapted viruses but, most notably, was also a feature of the 1983 North American outbreak of H5N2 highly pathogenic avian influenza virus. In identifying a 14th influenza A polypeptide, our data reinforce the unexpectedly high coding capacity of the viral genome and have implications for virus evolution, as well as for understanding the role of M2 in the virus life cycle.

Evolutionary conservation of the PA-X open reading frame in segment 3 of influenza A virus.

PA-X is a fusion protein of influenza A virus encoded in part from a +1 frameshifted X open reading frame (X-ORF) in segment 3. We show that the X-ORFs of diverse influenza A viruses can be divided into two groups that differ in selection pressure and likely function, reflected in the presence of an internal stop codon and a change in synonymous diversity. Notably, truncated forms of PA-X evolved convergently in swine and dogs, suggesting a strong species-specific effect.

Analysis by single-gene reassortment demonstrates that the 1918 influenza virus is functionally compatible with a low-pathogenicity avian influenza virus in mice.

The 1918-1919 "Spanish" influenza pandemic is estimated to have caused 50 million deaths worldwide. Understanding the origin, virulence, and pathogenic properties of past pandemic influenza viruses, including the 1918 virus, is crucial for current public health preparedness and future pandemic planning. The origin of the 1918 pandemic virus has not been resolved, but its coding sequences are very like those of avian influenza virus. The proteins encoded by the 1918 virus differ from typical low-pathogenicity avian influenza viruses at only a small number of amino acids in each open reading frame. In this study, a series of chimeric 1918 influenza viruses were created in which each of the eight 1918 pandemic virus gene segments was replaced individually with the corresponding gene segment of a prototypical low-pathogenicity avian influenza (LPAI) H1N1 virus in order to investigate functional compatibility of the 1918 virus genome with gene segments from an LPAI virus and to identify gene segments and mutations important for mammalian adaptation. This set of eight "7:1" chimeric viruses was compared to the parental 1918 and LPAI H1N1 viruses in intranasally infected mice. Seven of the 1918 LPAI 7:1 chimeric viruses replicated and caused disease equivalent to the fully reconstructed 1918 virus. Only the chimeric 1918 virus containing the avian influenza PB2 gene segment was attenuated in mice. This attenuation could be corrected by the single E627K amino acid change, further confirming the importance of this change in mammalian adaptation and mouse pathogenicity. While the mechanisms of influenza virus host switch, and particularly mammalian host adaptation are still only partly understood, these data suggest that the 1918 virus, whatever its origin, is very similar to avian influenza virus.

Overlapping signals for translational regulation and packaging of influenza A virus segment 2.

Influenza A virus segment 2 mRNA expresses three polypeptides: PB1, PB1-F2 and PB1-N40, from AUGs 1, 4 and 5 respectively. Two short open reading frames (sORFs) initiated by AUGs 2 and 3 are also present. To understand translational regulation in this system, we systematically mutated AUGs 1-4 and monitored polypeptide synthesis from plasmids and recombinant viruses. This identified sORF2 as a key regulatory element with opposing effects on PB1-F2 and PB1-N40 expression. We propose a model in which AUGs 1-4 are accessed by leaky ribosomal scanning, with sORF2 repressing synthesis of downstream PB1-F2. However, sORF2 also up-regulates PB1-N40 expression, most likely by a reinitiation mechanism that permits skipping of AUG4. Surprisingly, we also found that in contrast to plasmid-driven expression, viruses with improved AUG1 initiation contexts produced less PB1 in infected cells and replicated poorly, producing virions with elevated particle:PFU ratios. Analysis of the genome content of virus particles showed reduced packaging of the mutant segment 2 vRNAs. Overall, we conclude that segment 2 mRNA translation is regulated by a combination of leaky ribosomal scanning and reinitiation, and that the sequences surrounding the PB1 AUG codon are multifunctional, containing overlapping signals for translation initiation and for segment-specific packaging.

Clinical Characteristics of the 2019 Eastern Equine Encephalitis Outbreak in Michigan.

Background: Eastern equine encephalitis virus is a mosquito-borne alphavirus responsible for unpredictable outbreaks of severe neurologic disease in animals and humans. While most human infections are asymptomatic or clinically nonspecific, a minority of patients develops encephalitic disease, a devastating illness with a mortality rate of ≥30%. No treatments are known to be effective. Eastern equine encephalitis virus infection is rare in the United States, with an annual average nationwide incidence of 7 cases between 2009 and 2018. However, in 2019, 38 cases were confirmed nationwide, including 10 in Michigan. Methods: Data from 8 cases identified by a regional network of physicians in southwest Michigan were abstracted from clinical records. Clinical imaging and histopathology were aggregated and reviewed. Results: Patients were predominantly older adults (median age, 64 years), and all were male. Results of initial arboviral cerebrospinal fluid serology were frequently negative, and diagnosis was not made until a median of 24.5 days (range, 13-38 days) after presentation, despite prompt lumbar punctures in all patients. Imaging findings were dynamic and heterogeneous, with abnormalities of the thalamus and/or basal ganglia, and prominent pons and midbrain abnormalities were displayed in 1 patient. Six patients died, 1 survived the acute illness with severe neurologic sequelae, and 1 recovered with mild sequelae. A limited postmortem examination revealed diffuse meningoencephalitis, neuronophagia, and focal vascular necrosis. Conclusions: Eastern equine encephalitis is a frequently fatal condition whose diagnosis is often delayed, and for which no effective treatments are known. Improved diagnostics are needed to facilitate patient care and encourage the development of treatments.

Rapid selection of a transmissible multidrug-resistant influenza A/H3N2 virus in an immunocompromised host.

BACKGROUND: The overall impact of influenza virus infection in immunocompromised patients is largely unknown. Antigenic drift and genetic variations during prolonged influenza infection have been demonstrated. In this report we describe a multidrug-resistant H3N2 influenza virus isolated from an immunocompromised patient after 5 days of therapy. METHODS: Multiple nasal wash samples were collected from an infected patient, and viral isolates were characterized. Sensitivity to antiviral agents was evaluated. Fitness and transmissibility were assessed in ferrets and tissue culture. RESULTS: An in-frame 4-amino acid deletion emerged in the neuraminidase (NA) gene of an H3N2 virus after 5 days of oseltamivir therapy. No other changes in the NA or hemagglutinin genes were noted. Drug sensitivity assays revealed resistance to oseltamivir (>10-fold increase in 50% inhibitory concentration [IC(50)]) and reduction in sensitivity to zanamivir (3-7-fold increase in IC(50) or 50% effective concentration). No change in fitness or transmissibility was observed. CONCLUSIONS: An in-frame NA gene deletion was rapidly selected for in an immunocompromised patient, resulting in decreased sensitivity of the isolate to available NA inhibitors without a change in fitness or transmissibility. This finding has implications for our understanding of the emergence of antiviral resistance and treatment of patients with influenza A infection, especially those who are immunocompromised.

Bilateral peripheral facial paralysis during pregnancy: a presentation of acute HIV seroconversion.

A G7P5A1 woman in her 40s presented to the emergency department at 37 weeks 3 days' estimated gestational age (EGA) with headache, lip tingling and several days of difficulty speaking. Physical examination demonstrated bilateral facial weakness in a peripheral distribution, as well as decreased corneal reflexes and cervical lymphadenopathy. Routine fourth generation HIV screening had previously been negative at 14 and 28 weeks' EGA. Brain MRI was unremarkable, and lumbar puncture disclosed a low-grade, mononuclear cerebrospinal fluid pleocytosis; the patient was treated supportively. She returned for induction of labour at 39 weeks, at which time HIV infection was unexpectedly diagnosed. While unilateral idiopathic peripheral facial paralysis is associated with the third trimester of pregnancy and the early postpartum period, bilateral facial paralysis is rare and should prompt work-up for an underlying systemic cause, such as HIV infection.

Recent human influenza A/H3N2 virus evolution driven by novel selection factors in addition to antigenic drift.

BACKGROUND: Examination of the evolutionary dynamics of complete influenza viral genomes reveals that other processes, in conjunction with antigenic drift, play important roles in viral evolution and selection, but there is little biological evidence to support these genomic data. Previous work demonstrated that after the A/Fujian/411/2002-like H3N2 influenza A epidemic during 2003-2004, a preexisting nondominant Fujian-like viral clade gained a small number of changes in genes encoding the viral polymerase complex, along with several changes in the antigenic regions of hemagglutinin, and in a genome-wide selective sweep, it replaced other co-circulating H3N2 clades. METHODS: Representative strains of these virus clades were evaluated in vitro and in vivo. RESULTS: The newly dominant 2004-2005 A/California/7/2004-like H3N2 clade, which featured 2 key amino acid changes in the polymerase PA segment, grew to higher titers in MDCK cells and ferret tissues and caused more-severe disease in ferrets. The polymerase complex of this virus demonstrated enhanced activity in vitro, correlating directly to the enhanced replicative fitness and virulence in vivo. CONCLUSION: These data suggest that influenza strains can be selected in humans through mutations that increase replicative fitness and virulence, in addition to the well-characterized antigenic changes in the surface glycoproteins.

Encephalitis with coinfection by Jamestown canyon virus (JCV) and varicella zoster virus (VZV).

We present the case of a 59-year-old Midwestern farmer who presented with altered mental status, dysarthria, urinary incontinence, and a right-sided L5 dermatomal rash; he had recently received a course of oral corticosteroids for treatment of radicular low back pain. Lumbar puncture revealed the presence of varicella zoster virus (VZV) and IgM antibodies against a California-group encephalitis virus, later confirmed as Jamestown Canyon virus (JCV). Unfortunately, the patient's health declined despite aggressive treatment, developing progressive subarachnoid hemorrhage. He died after withdrawal of supportive care following 3 weeks in the intensive care unit. To our knowledge, this is the first documented case of encephalitis associated with coinfection by VZV and JCV. While the relative contributions of these viral pathogens to the patient's illness are difficult to ascertain, the clinical features of this case are consistent with co-pathogenesis, possibly driven by antecedent corticosteroid use. This case highlights the emerging role of viral coinfections in the etiology of viral illnesses.

Dengue and Zika Virus Cross-Reactive Human Monoclonal Antibodies Protect against Spondweni Virus Infection and Pathogenesis in Mice.

Spondweni virus (SPOV) is the flavivirus that is most closely related to Zika virus (ZIKV). Although SPOV causes sporadic human infections in Africa, recently it was found in Culex mosquitoes in Haiti. To investigate the pathogenic spectrum of SPOV, we developed infection models in mice. Although two SPOV strains failed to cause disease in immunocompetent mice, each accumulated in the brain, spleen, eye, testis, and kidney when type I interferon signaling was blocked and unexpectedly caused infection, immune cell infiltration, and swelling in the ankle. In pregnant mice, SPOV replicated in the placenta and fetus but did not cause placental insufficiency or microcephaly. We identified human antibodies from ZIKV or DENV immune subjects that neutralized SPOV infection and protected against lethal challenge. Our experiments describe similarities and differences in clinical syndromes between SPOV and ZIKV and suggest that their serological relatedness has implications for antibody therapeutics and flavivirus vaccine development.

Animal Models of Zika Virus Infection during Pregnancy.

Zika virus (ZIKV) emerged suddenly in the Americas in 2015 and was associated with a widespread outbreak of microcephaly and other severe congenital abnormalities in infants born to mothers infected during pregnancy. Vertical transmission of ZIKV in humans was confirmed when viral RNA was detected in fetal and placental tissues, and this outcome has been recapitulated experimentally in animals. Unlike other flaviviruses, ZIKV is both arthropod- and sexually-transmitted, and has a broad tissue tropism in humans, including multiple tissues of the reproductive tract. The threats posed by ZIKV have prompted the development of multiple in vivo models to better understand the pathogenesis of ZIKV, particularly during pregnancy. Here, we review the progress on animal models of ZIKV infection during pregnancy. These studies have generated a foundation of insights into the biology of ZIKV, and provide a means for evaluating vaccines and therapeutics.

Gestational Stage and IFN-λ Signaling Regulate ZIKV Infection In Utero.

Although Zika virus (ZIKV)-induced congenital disease occurs more frequently during early stages of pregnancy, its basis remains undefined. Using established type I interferon (IFN)-deficient mouse models of ZIKV transmission in utero, we found that the placenta and fetus were more susceptible to ZIKV infection at earlier gestational stages. Whereas ZIKV infection at embryonic day 6 (E6) resulted in placental insufficiency and fetal demise, infections at midstage (E9) resulted in reduced cranial dimensions, and infection later in pregnancy (E12) caused no apparent fetal disease. In addition, we found that fetuses lacking type III IFN-λ signaling had increased ZIKV replication in the placenta and fetus when infected at E12, and reciprocally, treatment of pregnant mice with IFN-λ2 reduced ZIKV infection. IFN-λ treatment analogously diminished ZIKV infection in human midgestation fetal- and maternal-derived tissue explants. Our data establish a model of gestational stage dependence of ZIKV pathogenesis and IFN-λ-mediated immunity at the maternal-fetal interface.