Building a global immune system.

The COVID-19 pandemic exposed global vulnerabilities to emerging infectious diseases, heralded by earlier outbreaks, that did not result in appropriate investments in surveillance, international collaboration, and response. We propose specific steps that should be taken to reduce future risks to public health, economic and political stability, and food security.

Prenatal fever and autism risk.

Some studies suggest that prenatal infection increases risk of autism spectrum disorders (ASDs). This study was undertaken in a prospective cohort in Norway to examine whether we could find evidence to support an association of the prenatal occurrence of fever, a common manifestation of infection, with ASD risk. Prospective questionnaires provided maternal exposure data; case status was established from clinical assessments and registry linkages. In a large, prospectively ascertained cohort of pregnant mothers and their offspring, we examined infants born ⩾32 weeks for associations between fever exposure in each trimester and ASD risk using logistic regression. Maternal exposure to second-trimester fever was associated with increased ASD risk, adjusting for presence of fever in other trimesters and confounders (adjusted odds ratio (aOR), 1.40; 95% confidence interval, 1.09-1.79), with a similar, but nonsignificant, point estimate in the first trimester. Risk increased markedly with exposure to three or more fever episodes after 12 weeks' gestation (aOR, 3.12; 1.28-7.63). ASD risk appears to increase with maternal fever, particularly in the second trimester. Risk magnified dose dependently with exposure to multiple fevers after 12 weeks' gestation. Our findings support a role for gestational maternal infection and innate immune responses to infection in the pathogenesis of at least some cases of ASD.

Global patterns in coronavirus diversity.

Since the emergence of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrom Coronavirus (MERS-CoV) it has become increasingly clear that bats are important reservoirs of CoVs. Despite this, only 6% of all CoV sequences in GenBank are from bats. The remaining 94% largely consist of known pathogens of public health or agricultural significance, indicating that current research effort is heavily biased towards describing known diseases rather than the 'pre-emergent' diversity in bats. Our study addresses this critical gap, and focuses on resource poor countries where the risk of zoonotic emergence is believed to be highest. We surveyed the diversity of CoVs in multiple host taxa from twenty countries to explore the factors driving viral diversity at a global scale. We identified sequences representing 100 discrete phylogenetic clusters, ninety-one of which were found in bats, and used ecological and epidemiologic analyses to show that patterns of CoV diversity correlate with those of bat diversity. This cements bats as the major evolutionary reservoirs and ecological drivers of CoV diversity. Co-phylogenetic reconciliation analysis was also used to show that host switching has contributed to CoV evolution, and a preliminary analysis suggests that regional variation exists in the dynamics of this process. Overall our study represents a model for exploring global viral diversity and advances our fundamental understanding of CoV biodiversity and the potential risk factors associated with zoonotic emergence.

Immune network analysis of cerebrospinal fluid in myalgic encephalomyelitis/chronic fatigue syndrome with atypical and classical presentations.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a persistent and debilitating disorder marked by cognitive and sensory dysfunction and unexplained physical fatigue. Classically, cases present after a prodrome consistent with infection; however, some cases are atypical and have a different presentation and comorbidities that pose challenges for differential diagnosis. We analyzed cerebrospinal fluid (CSF) from 32 cases with classical ME/CFS and 27 cases with atypical ME/CFS using a 51-plex cytokine assay. Atypical subjects differed in cytokine profiles from classical subjects. In logistic regression models incorporating immune molecules that were identified as potential predictor variables through feature selection, we found strong associations between the atypical ME/CFS phenotype and lower CSF levels of the inflammatory mediators, interleukin 17A and CXCL9. Network analysis revealed an absence of inverse inter-cytokine relationships in CSF from atypical patients, and more sparse positive intercorrelations, than classical subjects. Interleukin 1 receptor antagonist appeared to be a negative regulator in classical ME/CFS, with patterns suggestive of disturbances in interleukin 1 signaling and autoimmunity-type patterns of immune activation. Immune signatures in the central nervous system of ME/CFS patients with atypical features may be distinct from those with more typical clinical presentations.

Further Evidence for Bats as the Evolutionary Source of Middle East Respiratory Syndrome Coronavirus.

The evolutionary origins of Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) are unknown. Current evidence suggests that insectivorous bats are likely to be the original source, as several 2c CoVs have been described from various species in the family Vespertilionidae Here, we describe a MERS-like CoV identified from a Pipistrellus cf. hesperidus bat sampled in Uganda (strain PREDICT/PDF-2180), further supporting the hypothesis that bats are the evolutionary source of MERS-CoV. Phylogenetic analysis showed that PREDICT/PDF-2180 is closely related to MERS-CoV across much of its genome, consistent with a common ancestry; however, the spike protein was highly divergent (46% amino acid identity), suggesting that the two viruses may have different receptor binding properties. Indeed, several amino acid substitutions were identified in key binding residues that were predicted to block PREDICT/PDF-2180 from attaching to the MERS-CoV DPP4 receptor. To experimentally test this hypothesis, an infectious MERS-CoV clone expressing the PREDICT/PDF-2180 spike protein was generated. Recombinant viruses derived from the clone were replication competent but unable to spread and establish new infections in Vero cells or primary human airway epithelial cells. Our findings suggest that PREDICT/PDF-2180 is unlikely to pose a zoonotic threat. Recombination in the S1 subunit of the spike gene was identified as the primary mechanism driving variation in the spike phenotype and was likely one of the critical steps in the evolution and emergence of MERS-CoV in humans.IMPORTANCE Global surveillance efforts for undiscovered viruses are an important component of pandemic prevention initiatives. These surveys can be useful for finding novel viruses and for gaining insights into the ecological and evolutionary factors driving viral diversity; however, finding a viral sequence is not sufficient to determine whether it can infect people (i.e., poses a zoonotic threat). Here, we investigated the specific zoonotic risk of a MERS-like coronavirus (PREDICT/PDF-2180) identified in a bat from Uganda and showed that, despite being closely related to MERS-CoV, it is unlikely to pose a threat to humans. We suggest that this approach constitutes an appropriate strategy for beginning to determine the zoonotic potential of wildlife viruses. By showing that PREDICT/PDF-2180 does not infect cells that express the functional receptor for MERS-CoV, we further show that recombination was likely to be the critical step that allowed MERS to emerge in humans.

Syncytial Hepatitis of Tilapia ( Oreochromis niloticus L.) is Associated With Orthomyxovirus-Like Virions in Hepatocytes.

Using transmission electron microscopy (TEM), the presented work expands on the ultrastructural findings of an earlier report on "syncytial hepatitis," a novel disease of tilapia (SHT). Briefly, TEM confirmed the presence of an orthomyxovirus-like virus within the diseased hepatocytes but not within the endothelium. This was supported by observing extracellular and intracellular (mostly intraendosomal), 60-100 nm round virions with a trilaminar capsid containing up to 7 electron-dense aggregates. Other patterns noted included enveloped or filamentous virions and virion-containing cytoplasmic membrane folds, suggestive of endocytosis. Patterns atypical for orthymyxovirus included the formation of syncytia and the presence of virions within the perinuclear cisternae (suspected to be the Golgi apparatus). The ultrastructural morphology of SHT-associated virions is similar to that previously reported for tilapia lake virus (TiLV). A genetic homology was investigated using the available reverse transcriptase polymerase chain reaction (RT-PCR) probes for TiLV and comparing clinically sick with clinically normal fish and negative controls. By RT-PCR analysis, viral nucleic acid was detected only in diseased fish. Taken together, these findings strongly suggest that a virus is causally associated with SHT, that this virus shares ultrastructural features with orthomyxoviruses, and it presents with partial genetic homology with TiLV (190 nucleotides).

Cytokine network analysis of cerebrospinal fluid in myalgic encephalomyelitis/chronic fatigue syndrome.

Myalgic encephalomyelitis/chronic fatigue syndrome is an unexplained debilitating disorder that is frequently associated with cognitive and motor dysfunction. We analyzed cerebrospinal fluid from 32 cases, 40 subjects with multiple sclerosis and 19 normal subjects frequency-matched for age and sex using a 51-plex cytokine assay. Group-specific differences were found for the majority of analytes with an increase in cases of CCL11 (eotaxin), a chemokine involved in eosinophil recruitment. Network analysis revealed an inverse relationship between interleukin 1 receptor antagonist and colony-stimulating factor 1, colony-stimulating factor 2 and interleukin 17F, without effects on interleukin 1α or interleukin 1ß, suggesting a disturbance in interleukin 1 signaling. Our results indicate a markedly disturbed immune signature in the cerebrospinal fluid of cases that is consistent with immune activation in the central nervous system, and a shift toward an allergic or T helper type-2 pattern associated with autoimmunity.

Preliminary Survey of Ectoparasites and Associated Pathogens from Norway Rats in New York City.

The Norway rat (Rattus norvegicus) is a reservoir of many zoonotic pathogens and lives in close proximity to humans in urban environments. Human infection with rodent-borne disease occurs either directly through contact with a rat or its excreta, or indirectly via arthropod vectors such as fleas and ticks. Here, we report on the diversity and abundance of ectoparasitic arthropod species and associated pathogenic bacteria from 133 Norway rats trapped over a 10-mo period in Manhattan, New York, NY. Norway rats were host to the tropical rat mite [Ornithonyssus bacoti (Hirst)], the spiny rat mite (Laelaps echidnina Berlese), Laelaps nuttalli Hirst, the spined rat louse [Polyplax spinulosa (Burmeister)], and the Oriental rat flea [(Xenopsylla cheopis) (Rothschild)], with an average of 1.7 species per individual. A flea index of 4.1 X. cheopis was determined, whereas previous studies in New York City reported 0.22 fleas per rat. Multiple species of pathogenic Bartonella were identified from Oriental rat fleas that were related to Bartonella tribocorum, Bartonella rochalimae, and Bartonella elizabethae. However, no evidence of Yersinia pestis or Rickettsia spp. infection was detected in fleas. The identification of multiple medically important ectoparasite species in New York City underscores the need for future efforts to fully characterize the diversity and distribution of ectoparasites on Norway rats, and assess the risk to humans of vector-borne disease transmission.

Discovery of a Novel Hepatovirus (Phopivirus of Seals) Related to Human Hepatitis A Virus.

UNLABELLED: Describing the viral diversity of wildlife can provide interesting and useful insights into the natural history of established human pathogens. In this study, we describe a previously unknown picornavirus in harbor seals (tentatively named phopivirus) that is related to human hepatitis A virus (HAV). We show that phopivirus shares several genetic and phenotypic characteristics with HAV, including phylogenetic relatedness across the genome, a specific and seemingly quiescent tropism for hepatocytes, structural conservation in a key functional region of the type III internal ribosomal entry site (IRES), and a codon usage bias consistent with that of HAV. IMPORTANCE: Hepatitis A virus (HAV) is an important viral hepatitis in humans because of the substantial number of cases each year in regions with low socioeconomic status. The origin of HAV is unknown, and no nonprimate HAV-like viruses have been described. Here, we describe the discovery of an HAV-like virus in seals. This finding suggests that the diversity and evolutionary history of these viruses might be far greater than previously thought and may provide insight into the origin and pathogenicity of HAV.

West Nile virus in the British Virgin Islands.

West Nile virus (WNV) first emerged in the US in 1999 and has since spread across the Americas. Here, we report the continued expansion of WNV to the British Virgin Islands following its emergence in a flock of free-roaming flamingos. Histologic review of a single chick revealed lesions consistent with WNV infection, subsequently confirmed with PCR, immunohistochemistry and in situ hybridization. Full genome analysis revealed 99% sequence homology to strains circulating in the US over the past decade. This study highlights the need for rapid necropsy of wild bird carcasses to fully understand the impact of WNV on wild populations.

Coronaviruses in bats from Mexico.

Bats are reservoirs for a wide range of human pathogens including Nipah, Hendra, rabies, Ebola, Marburg and severe acute respiratory syndrome coronavirus (CoV). The recent implication of a novel beta (ß)-CoV as the cause of fatal respiratory disease in the Middle East emphasizes the importance of surveillance for CoVs that have potential to move from bats into the human population. In a screen of 606 bats from 42 different species in Campeche, Chiapas and Mexico City we identified 13 distinct CoVs. Nine were alpha (α)-CoVs; four were ß-CoVs. Twelve were novel. Analyses of these viruses in the context of their hosts and ecological habitat indicated that host species is a strong selective driver in CoV evolution, even in allopatric populations separated by significant geographical distance; and that a single species/genus of bat can contain multiple CoVs. A ß-CoV with 96.5 % amino acid identity to the ß-CoV associated with human disease in the Middle East was found in a Nyctinomops laticaudatus bat, suggesting that efforts to identify the viral reservoir should include surveillance of the bat families Molossidae/Vespertilionidae, or the closely related Nycteridae/Emballonuridae. While it is important to investigate unknown viral diversity in bats, it is also important to remember that the majority of viruses they carry will not pose any clinical risk, and bats should not be stigmatized ubiquitously as significant threats to public health.

Emergence of fatal avian influenza in New England harbor seals.

UNLABELLED: From September to December 2011, 162 New England harbor seals died in an outbreak of pneumonia. Sequence analysis of postmortem samples revealed the presence of an avian H3N8 influenza A virus, similar to a virus circulating in North American waterfowl since at least 2002 but with mutations that indicate recent adaption to mammalian hosts. These include a D701N mutation in the viral PB2 protein, previously reported in highly pathogenic H5N1 avian influenza viruses infecting people. Lectin staining and agglutination assays indicated the presence of the avian-preferred SAα-2,3 and mammalian SAα-2,6 receptors in seal respiratory tract, and the ability of the virus to agglutinate erythrocytes bearing either the SAα-2,3 or the SAα-2,6 receptor. The emergence of this A/harbor seal/Massachusetts/1/2011 virus may herald the appearance of an H3N8 influenza clade with potential for persistence and cross-species transmission. IMPORTANCE: The emergence of new strains of influenza virus is always of great public concern, especially when the infection of a new mammalian host has the potential to result in a widespread outbreak of disease. Here we report the emergence of an avian influenza virus (H3N8) in New England harbor seals which caused an outbreak of pneumonia and contributed to a U.S. federally recognized unusual mortality event (UME). This outbreak is particularly significant, not only because of the disease it caused in seals but also because the virus has naturally acquired mutations that are known to increase transmissibility and virulence in mammals. Monitoring the spillover and adaptation of avian viruses in mammalian species is critically important if we are to understand the factors that lead to both epizootic and zoonotic emergence.

Absence of evidence for bornavirus infection in schizophrenia, bipolar disorder and major depressive disorder.

In 1983, reports of antibodies in subjects with major depressive disorder (MDD) to an as-yet uncharacterized infectious agent associated with meningoencephalitis in horses and sheep led to molecular cloning of the genome of a novel, negative-stranded neurotropic virus, Borna disease virus (BDV). This advance has enabled the development of new diagnostic assays, including in situ hybridization, PCR and serology based on recombinant proteins. Since these assays were first implemented in 1990, more than 80 studies have reported an association between BDV and a wide range of human illnesses that include MDD, bipolar disorder (BD), schizophrenia (SZ), anxiety disorder, chronic fatigue syndrome, multiple sclerosis, amyotrophic lateral sclerosis, dementia and glioblastoma multiforme. However, to date there has been no blinded case-control study of the epidemiology of BDV infection. Here, in a United States-based, multi-center, yoked case-control study with standardized methods for clinical assessment and blinded serological and molecular analysis, we report the absence of association of psychiatric illness with antibodies to BDV or with BDV nucleic acids in serially collected serum and white blood cell samples from 396 subjects, a study population comprised of 198 matched pairs of patients and healthy controls (52 SZ/control pairs, 66 BD/control pairs and 80 MDD/control pairs). Our results argue strongly against a role for BDV in the pathogenesis of these psychiatric disorders.

Genome-scale phylogeny of the alphavirus genus suggests a marine origin.

The genus Alphavirus comprises a diverse group of viruses, including some that cause severe disease. Using full-length sequences of all known alphaviruses, we produced a robust and comprehensive phylogeny of the Alphavirus genus, presenting a more complete evolutionary history of these viruses compared to previous studies based on partial sequences. Our phylogeny suggests the origin of the alphaviruses occurred in the southern oceans and spread equally through the Old and New World. Since lice appear to be involved in aquatic alphavirus transmission, it is possible that we are missing a louse-borne branch of the alphaviruses. Complete genome sequencing of all members of the genus also revealed conserved residues forming the structural basis of the E1 and E2 protein dimers.

A novel herpesvirus in 3 species of pheasants: mountain peacock pheasant (Polyplectron inopinatum), Malayan peacock pheasant (Polyplectron malacense), and Congo peafowl (Afropavo congensis).

The mountain peacock pheasant (Polyplectron inopinatum), the Malayan peacock pheasant (Polyplectron malacense), and the Congo peafowl (Afropavo congensis) are all listed as vulnerable to extinction under the International Union for Conservation of Nature Red List of Threatened Species. Here the authors report fatal infection with a novel herpesvirus in all 3 species of birds. DNA was extracted from the livers of birds with hepatocellular necrosis and intranuclear eosinophilic inclusions consistent with herpesvirus infection. Based on degenerate herpesvirus primers and polymerase chain reaction, 220- and 519-base pair products of the herpes DNA polymerase and DNA terminase genes, respectively, were amplified. Sequence analysis revealed that all birds were likely infected with the same virus. At the nucleotide level, the pheasant herpesvirus had 92% identity with gallid herpesvirus 3 and 77.7% identity with gallid herpesvirus 2. At the amino acid level, the herpes virus had 93.8% identity with gallid herpesvirus 3 and 89.4% identity with gallid herpesvirus 2. These findings indicate that the closest relative to this novel herpesvirus is gallid herpesvirus 3, a nonpathogenic virus used widely in a vaccine against Marek's disease. In situ hybridization using probes specific to the peacock pheasant herpesvirus DNA polymerase revealed strong intranuclear staining in the necrotic liver lesions of an infected Malayan peacock pheasant but no staining in normal liver from an uninfected bird. The phasianid herpesvirus reported here is a novel member of the genus Mardivirus of the subfamily Alphaherpesvirinae and is distinct from other galliform herpesviruses.

Characterization of Durham virus, a novel rhabdovirus that encodes both a C and SH protein.

The family Rhabdoviridae is a diverse group of non-segmented, negative-sense RNA viruses that are distributed worldwide and infect a wide range of hosts including vertebrates, invertebrates, and plants. Of the 114 currently recognized vertebrate rhabdoviruses, relatively few have been well characterized at both the antigenic and genetic level; hence, the phylogenetic relationships between many of the vertebrate rhabdoviruses remain unknown. The present report describes a novel rhabdovirus isolated from the brain of a moribund American coot (Fulica americana) that exhibited neurological signs when found in Durham County, North Carolina, in 2005. Antigenic characterization of the virus revealed that it was serologically unrelated to 68 other known vertebrate rhabdoviruses. Genomic sequencing of the virus indicated that it shared the highest identity to Tupaia rhabdovirus (TUPV), and as only previously observed in TUPV, the genome encoded a putative C protein in an overlapping open reading frame (ORF) of the phosphoprotein gene and a small hydrophobic (SH) protein located in a novel ORF between the matrix and glycoprotein genes. Phylogenetic analysis of partial amino acid sequences of the nucleoprotein and polymerase protein indicated that, in addition to TUPV, the virus was most closely related to avian and small mammal rhabdoviruses from Africa and North America. In this report, we present the morphological, pathological, antigenic, and genetic characterization of the new virus, tentatively named Durham virus (DURV), and discuss its potential evolutionary relationship to other vertebrate rhabdoviruses.

Use of nucleotide composition analysis to infer hosts for three novel picorna-like viruses.

Nearly complete genome sequences of three novel RNA viruses were acquired from the stool of an Afghan child. Phylogenetic analysis indicated that these viruses belong to the picorna-like virus superfamily. Because of their unique genomic organization and deep phylogenetic roots, we propose these viruses, provisionally named calhevirus, tetnovirus-1, and tetnovirus-2, as prototypes of new viral families. A newly developed nucleotide composition analysis (NCA) method was used to compare mononucleotide and dinucleotide frequencies for RNA viruses infecting mammals, plants, or insects. Using a large training data set of 284 representative picornavirus-like genomic sequences with defined host origins, NCA correctly identified the kingdom or phylum of the viral host for >95% of picorna-like viruses. NCA predicted an insect host origin for the 3 novel picorna-like viruses. Their presence in human stool therefore likely reflects ingestion of insect-contaminated food. As metagenomic analyses of different environments and organisms continue to yield highly divergent viral genomes NCA provides a rapid and robust method to identify their likely cellular hosts.

The Autism Birth Cohort: a paradigm for gene-environment-timing research.

The reported prevalence of autism spectrum disorders (ASDs) has increased by 5- to 10-fold over the past 20 years. Whether ASDs are truly more frequent is controversial; nonetheless, the burden is profound in human and economic terms. Although autism is among the most heritable of mental disorders, its pathogenesis remains obscure. Environmental factors are proposed; however, none is implicated. Furthermore, there are no biomarkers to screen for ASD or risk of ASD. The Autism Birth Cohort (ABC) was initiated to analyze gene x environment x timing interactions and enable early diagnosis. It uses a large, unselected birth cohort in which cases are prospectively ascertained through population screening. Samples collected serially through pregnancy and childhood include parental blood, maternal urine, cord blood, milk teeth and rectal swabs. More than 107,000 children are continuously screened through questionnaires, referral, and a national registry. Cases are compared with a control group from the same cohort in a 'nested case-control' design. Early screening and diagnostic assessments and re-assessments are designed to provide a rich view of longitudinal trajectory. Genetic, proteomic, immunologic, metagenomic and microbiological tools will be used to exploit unique biological samples. The ABC is a paradigm for analyzing the role of genetic and environmental factors in complex disorders.

Nipah virus outbreak with person-to-person transmission in a district of Bangladesh, 2007.

In February 2007 an outbreak of Nipah virus (NiV) encephalitis in Thakurgaon District of northwest Bangladesh affected seven people, three of whom died. All subsequent cases developed illness 7-14 days after close physical contact with the index case while he was ill. Cases were more likely than controls to have been in the same room (100% vs. 9.5%, OR undefined, P<0.001) and to have touched him (83% vs. 0%, OR undefined, P<0.001). Although the source of infection for the index case was not identified, 50% of Pteropus bats sampled from near the outbreak area 1 month after the outbreak had antibodies to NiV confirming the presence of the virus in the area. The outbreak was spread by person-to-person transmission. Risk of NiV infection in family caregivers highlights the need for infection control practices to limit transmission of potentially infectious body secretions.

Passive transfer of streptococcus-induced antibodies reproduces behavioral disturbances in a mouse model of pediatric autoimmune neuropsychiatric disorders associated with streptococcal infection.

Streptococcal infections can induce obsessive-compulsive and tic disorders. In children, this syndrome, frequently associated with disturbances in attention, learning and mood, has been designated pediatric autoimmune neuropsychiatric disorders associated with streptococcal infection (PANDAS). Autoantibodies recognizing central nervous system (CNS) epitopes are found in sera of most PANDAS subjects, but may not be unique to this neuropsychiatric subset. In support of a humoral immune mechanism, clinical improvement often follows plasmapheresis or intravenous immunoglobulin. We recently described a PANDAS mouse model wherein repetitive behaviors correlate with peripheral anti-CNS antibodies and immune deposits in brain following streptococcal immunization. These antibodies are directed against group A beta-hemolytic streptococcus matrix (M) protein and cross-react with molecular targets complement C4 protein and alpha-2-macroglobulin in brain. Here we show additional deficits in motor coordination, learning/memory and social interaction in PANDAS mice, replicating more complex aspects of human disease. Furthermore, we demonstrate for the first time that humoral immunity is necessary and sufficient to induce the syndrome through experiments wherein naive mice are transfused with immunoglobulin G (IgG) from PANDAS mice. Depletion of IgG from donor sera abrogates behavior changes. These functional disturbances link to the autoimmunity-related IgG1 subclass but are not attributable to differences in cytokine profiles. The mode of disrupting blood-brain barrier integrity differentially affects the ultimate CNS distribution of these antibodies and is shown to be an additional important determinant of neuropsychiatric outcomes. This work provides insights into PANDAS pathogenesis and may lead to new strategies for identification and treatment of children at risk for autoimmune brain disorders.