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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.

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 1a 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.

ABSTRACTThe 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.

ABSTRACTHere we propose the use of a 216-nucleotide fragment located in the carboxyl terminus of the E gene (E-COOH) and a pairwise-based comparison method for genotyping of dengue virus 1 (DENV-1) strains. We have applied this method to the detection and characterization of DENV-1 in serum samples from travelers returning from the tropics. The results obtained with the typing system correlate with the results obtained by comparison of the sequences of the entire E gene of the strains. The approach demonstrates utility in plotting the distribution and circulation of different genotypes of DENV-1 and also suggests the presence of two new clades of Indian strains. The integration of the method with an online database and a typing characterization tool enhances its strength. Additionally, the analysis of the complete E gene of DENV-1 strains suggested the occurrence of a nondescribed recombination event in the China GD23-95 strain. We propose the use of this methodology as a tool for real-time epidemiological surveillance of dengue virus infections and their pathogenesis.

Here we propose the use of a 216-nucleotide fragment located in the carboxyl terminus of the E gene (E-COOH) and a pairwise-based comparison method for genotyping of dengue virus 1 (DENV-1) strains. We have applied this method to the detection and characterization of DENV-1 in serum samples from travelers returning from the tropics. The results obtained with the typing system correlate with the results obtained by comparison of the sequences of the entire E gene of the strains. The approach demonstrates utility in plotting the distribution and circulation of different genotypes of DENV-1 and also suggests the presence of two new clades of Indian strains. The integration of the method with an online database and a typing characterization tool enhances its strength. Additionally, the analysis of the complete E gene of DENV-1 strains suggested the occurrence of a nondescribed recombination event in the China GD23-95 strain. We propose the use of this methodology as a tool for real-time epidemiological surveillance of dengue virus infections and their pathogenesis.

ABSTRACTHere we present a system for adenovirus detection and genotyping based on PCR amplification and phylogenetic analysis of a conserved hexon gene fragment. The system was validated using 157 sequences (86 previously typed and 71 clinical samples) and correctly identified species and serotype in 100% and 84% of sequences, respectively. Known associations between specific serotypes and clinical syndromes are verified. Possible new associations are described to allow further independent testing.

ABSTRACTA sensitive nested reverse transcription-PCR assay, targeting a short fragment of the gene encoding the small hydrophobic protein (SH gene), was developed to allow rapid characterization of mumps virus in clinical samples. The sensitivity and specificity of the assay were established using representative genotypes A, B, C, D, E, and F. Mumps virus RNA was characterized directly from cerebrospinal fluid (CSF) samples and in extracts of mumps virus isolates from patients with various clinical syndromes. Direct sequencing of products and subsequent phylogenetic analysis enabled genetic classification. A simple web-based system of sequence analysis was established. The study also allowed characterization of mumps virus strains from Argentina as part of a new subgenotype. This PCR assay for characterization of mumps infections coupled to a web-based analytical program provides a rapid method for identification of known and novel strains.

Borna disease virus is a nonsegmented negative-strand RNA virus that causes neurologic disease in a wide variety of animal hosts. Here we describe identification and characterization of the first glycoprotein in this viral system. The 18-kDa glycoprotein, gp18, has been purified from infected rat brain. Isolation and microsequencing of this protein allowed identification of a 16.2-kDa open reading frame in the viral antigenome. Lectin binding and endoglycosidase sensitivity assays indicate that gp18 is an unusual N-linked glycoprotein.

Borna disease virus (BDV) is a nonsegmented, negative-strand RNA virus that causes neurologic disorders in a wide range of animal species. Although the virus is unclassified, sequence analysis of the 8.9-kb viral genome has shown that it is related to rhabdoviruses and paramyxoviruses. We have mapped subgenomic RNAs of BDV strain He80-1 to the viral genome by determining the precise sequences at their 5' and 3' termini. This analysis showed that the genome contains three transcription initiation sites and four termination sites. A 14- to 16-nucleotide semiconserved sequence was present at the gene start sites and partially copied into the subgenomic RNAs. The termination sites contained a U-rich motif reminiscent of termination signals in rhabdoviruses and paramyxoviruses. In contrast to the genomes of other nonsegmented, negative-strand RNA viruses, the BDV genome lacked the typical configuration of termination signal, intergenic region, and initiation signal at the gene boundaries. Instead, transcription units and transcription signals frequently overlapped. These differences have implications for our understanding of the control of viral transcription and may relate to the low-level replication and persistence of BDV.

Borna disease virus (BDV) is a nonsegmented, negative-strand RNA virus related to rhabdoviruses and paramyxoviruses. Unlike animal viruses of these two families, BDV transcribes RNAs in the nuclei of infected cells and produces high levels of transcripts containing multiple open reading frames. Previous Northern blot analysis of RNA from BDV-infected rat brain tissue has shown that two viral transcripts, a 6.1-kb RNA and a 1.5-kb RNA, lack regions that are internal to two otherwise identical transcripts, the 7.1-kb RNA and the 2.8-kb RNA, respectively (T. Briese, A. Schneemann, A. Lewis, Y. Park, S. Kim, H. Ludwig, and W. I. Lipkin, Proc. Natl. Acad. Sci. USA 91:4362-4366, 1994). To determine the precise location of this deletion, we performed reverse transcription PCR analysis using total RNA from BDV-infected rat brain tissue. This investigation resulted in the identification of two introns in the 7.1- and 2.8-kb RNAs, which can be alternatively spliced to yield additional RNA species, including the 6.1- and 1.5-kb RNAs. Transient transfection of COS-7 cells with a cDNA clone of the 2.8-kb RNA resulted in the production of both the 2.8-kb RNA and the 1.5-kb RNA, confirming the theory that the 2.8-kb RNA is a sufficient substrate for splicing in mammalian cells. Splicing has not previously been observed in nonsegmented, negative-strand RNA viruses and presumably serves as a mechanism by which expression of BDV proteins is regulated in infected cells.

ABSTRACTBorna disease virus (BDV) is a negative-strand RNA virus that infects the central nervous systems (CNS) of warm-blooded animals and causes disturbances of movement and behavior. The basis for neurotropism remains poorly understood; however, the observation that the distribution of infectious virus in immunocompetent rats is different from that in immunoincompetent rats indicates a role for the immune system in BDV tropism: whereas in immunocompetent rats virus is restricted to the central, peripheral, and autonomic nervous systems, immunoincompetent rats also have virus in nonneural tissues. In an effort to examine the influence of the humoral immune response on BDV pathogenesis, we examined the effects of passive immunization with neutralizing antiserum in immunoincompetent rats. Serum transfer into immunoincompetent rats did not prevent persistent CNS infection but did result in restriction of virus to neural tissues. These results indicate that neutralizing antibodies may play a role in preventing generalized infection with BDV.

Borna disease virus (BDV), the prototype of a new family within the order Mononegavirales, is unusual in its nuclear localization for replication and transcription and use of RNA splicing for gene expression. The BDV antigenome contains three transcription units and six major open reading frames. Multicistronic RNAs containing two introns are elaborated from the third transcription unit. Differential splicing of the two introns and cytoplasmic accumulation of the unspliced and partially spliced RNA are critical for the balanced expression of the putative matrix protein, glycoprotein, and polymerase. To investigate the mechanisms for cytoplasmic expression of unspliced and partially spliced BDV transcripts, the levels of these transcripts were measured in the cytoplasm of infected COS-7 cells and noninfected COS-7 cells transfected with plasmids containing 2.8-kb cDNA inserts representing either wild-type or mutant BDV RNA from the third transcription unit. Analysis of truncation mutations allowed the identification of a cis-acting element present within the 3' end of the BDV 2.8-kb transcript that facilitated the cytoplasmic accumulation of unspliced BDV transcripts through nucleocytoplasmic transport. The nucleocytoplasmic transport activity was not dependent on the presence of BDV proteins. Gel-shift assays revealed that the cis-acting element binds specifically to host cytoplasmic and nuclear proteins.

Rabbits were immunized with synthetic peptides representing the neurotransmitter dodecapeptide somatostatin-28(1-12) (SANSNPAMAPRE) coupled to the carrier protein keyhole limpet hemocyanin (KLH) at either the amino or the carboxyl terminus. Although all rabbits produced high-titer antisera to immunizing peptide, as assayed by ELISA, only rabbits immunized with peptide coupled to carrier at the amino terminus yielded antibodies that bound to native somatostatin in mouse brain slices. This effect of peptide coupling orientation on epitope specificity of peptide antisera is likely to be significant to other investigators who use predetermined peptide sequences to generate immunohistochemical reagents.

Borna disease virus (BDV) is a newly classified nonsegmented negative-strand RNA virus (order of Mononegavirales) that persistently infects specific brain regions and circuits of warm-blooded animals to cause behavioral disturbances. Viruses within the order of Mononegavirales have phosphoproteins that typically serve as transcription factors and are modulated in functional activity through phosphorylation. To identify the kinases involved in BDV phosphoprotein (BDV-P) phosphorylation, in vitro phosphorylation assays were performed using recombinant phosphoprotein produced in Escherichia coli as substrate and cytoplasmic extracts from a rat glioma cell line (C6) or rat brain extracts as sources of kinase activity. These experiments revealed that BDV-P was phosphorylated predominantly by protein kinase C (PKC) and to a lesser extent by casein kinase II. Partial purification of the PKC from rat brain extract suggested that the BDV-P phosphorylating kinase is PKCepsilon. A role for PKC phosphorylation in vivo was confirmed by using the PKC-specific inhibitor GF109203X. Furthermore, peptide mapping studies indicated that BDV-P is phosphorylated at the same sites in vitro as it is in vivo. Mutational analysis identified Ser26 and Ser28 as sites for PKC phosphorylation and Ser70 and Ser86 as sites for casein kinase II phosphorylation. The anatomic distribution of PKCepsilon in the central nervous system may have implications for BDV neurotropism and pathogenesis.

Borna disease virus (BDV) causes persistent central nervous system infection and behavioral disturbances in warm-blooded animals. Protein interaction studies were pursued to gain insight into the functions of the putative nucleoprotein (N), phosphoprotein (P), atypical glycoprotein (gp18), and X protein (X) of BDV. Coimmunoprecipitation experiments indicated that N and P, and P and X, form complexes in infected cells. Two-hybrid analyses confirmed interactions between P and P, P and X, and P and N, but not between P and gp18, N and gp18, X and gp18, or X and N. Analysis of P truncation mutants identified three nonoverlapping regions important for oligomerization (amino acids (aa) 135-172), and binding to X (aa 33-115) or N (aa 197-201). Coexpression of X stimulated oligomerization of P but decreased N-P complex formation. Immunocytochemistry of transfected noninfected CHO cells demonstrated that the distribution of X is dependent upon the presence of P-X expressed alone was found predominantly in the cytoplasm whereas coexpression of X and P resulted in nuclear localization. Immunocytochemistry of infected cells revealed nuclear colocalization of P and X. Interactions of P, N, and X may have implications for regulation of BDV transcription/replication and ribonucleoprotein assembly.

Borna disease virus, an unclassified infectious agent, causes immune-mediated neurologic disease in a wide variety of animal hosts and may be involved in pathogenesis of selected neuropsychiatric diseases in man. Initial reports suggested that Borna disease virus is a single-stranded RNA virus. We describe here a method for isolation of viral particles that has allowed definitive identification of the genome as containing a negative-polarity RNA. Further, we show that the viral mRNAs are transcribed in the nucleus.

The Borna disease virus antigenome includes five major open reading frames (ORFs) which encode, from 5' to 3', the putative nucleoprotein (N), the phosphoprotein (P), the putative matrix protein (M), the major glycoprotein (G), and the RNA-dependent RNA polymerase (pol). Whereas the N and P ORFs are translated from monocistronic transcripts, the M, G, and pol ORFs are translated from polycistronic transcripts. Expression of the M, G, and pol ORFs is dependent upon differential splicing of two introns (intron 1, 94 nucleotides [nt]; intron 2, 1,294 nt). In vitro transcription-translation assays of wild-type and mutant sequences indicated that the G ORF is translated from an unspliced 2.8-kb RNA by leaky ribosomal scanning. Splicing of intron 1 enhances the translation of the G ORF by converting the M ORF into a 13-amino-acid minicistron, a structure that facilitates ribosomal reinitiation.

The Borna disease virus (BDV) antigenome is comprised of five major open reading frames (ORFs). Products have been reported only for ORFs I, II, and III, encoding N (p40), P (p24/p23), and M (gp18), respectively. ORF IV predicts a 57-kDa protein with several potential glycosylation sites. Analysis of radiolabeled extracts from BDV-infected C6 cells and BHK-21 cells transfected with a Semliki Forest virus vector that contains ORF IV demonstrated the presence of a 94-kDa protein (G protein) which was sensitive to tunicamycin, endoglycosidase F/N-glycosidase, and endoglycosidase H but not to O-glycosidase. Sera from BDV-infected rats detected the G protein and had neutralization activity that was reduced following immunoadsorption with the G protein. Preincubation of cells with the G protein interfered with BDV infectivity. This effect was enhanced by treatment of the G protein with the exoglycosidase alpha-mannosidase and reduced after subsequent treatment with N-acetyl-beta-D-glucosaminidase. In concert these findings indicate that ORF IV encodes a 94-kDa N-linked glycoprotein with extensive high mannose- and/or hybrid-type oligosaccharide modifications. The presence of neutralization epitopes on the G protein and its capacity to interfere with infectivity suggest that the G protein is important for viral entry.

Borna disease virus infection is diagnosed by the presence of serum antibodies reactive with the major viral proteins, p40 and p23. Although p40 and p23 are unrelated in amino acid sequence structure, cross-reactive antibodies are described. Protein fragments and synthetic peptides were analyzed to characterize the specificities of antibodies to p23. Epitope mapping revealed eight continuous epitopes accessible on the surface of a predicted structural model for the monomeric and the disulfide-linked dimeric forms of p23. None of these epitopes was reactive with antibodies to p40. Cross-reactivity with monospecific sera and monoclonal antibodies to p40 was found for one discontinuous epitope located at the amino terminus of p23.

Borna disease is a neurologic syndrome caused by infection with a nonsegmented, negative-strand RNA virus, Borna disease virus. Infected animals have antibodies to two soluble viral proteins, p40 and p23, and a membrane-associated viral glycoprotein, gp18. We examined the time course for the development of neutralization activity and the expression of antibodies to individual viral proteins in sera of infected rats. The appearance of neutralizing activity correlated with the development of immunoreactivity to gp18, but not p40 or p23. Monospecific and monoclonal antibodies to native gp18 and recombinant nonglycosylated gp18 were also found to have neutralizing activity and to immunoprecipitate viral particles or subparticles. These findings suggest that gp18 is likely to be present on the surface of the viral particles and is likely to contain epitopes important for virus neutralization.

Coding and noncoding sequences were analyzed from field and experimental isolates of Borna disease virus. For a 24-kDa protein, maximum divergence was 1.5% at the predicted amino acid level and 3.1% at the nucleotide level. For a 40-kDa protein, maximum divergence was 1.1% at the predicted amino acid level and 4.1% at the nucleotide level. The highest variability in sequence (10%) was found in a 40-nucleotide stretch of genomic RNA between coding sequences for the 40- and 24-kDa proteins. The degree of sequence conservation in these isolates, passaged in various host species in vivo and in vitro over a period of 64 years, is unusual for negative-strand RNA viruses.

We report the sequence of a Borna disease virus clone (pBDV-40) that encodes a 40-kDa protein (p40) found in the nuclei of infected cells. Comparative sequence analysis indicates that p40 is distantly similar to two different regions in the L-polymerase proteins encoded by paramyxoviruses and rhabdoviruses. The p40 sequence similarity indicates a previously undetected duplication in these viral polymerases. Phylogenetic reconstruction suggests that the gene that encodes p40 last shared a common ancestor with these viral polymerase genes prior to the duplication event. These findings support the hypothesis that Borna disease virus is a negative-strand RNA virus and suggest that p40 is involved in transcription and/or replication. The discovery of a duplication within the polymerase proteins of paramyxoviruses and rhabdoviruses has profound implications for the mapping of enzymatic activities within these multifunctional proteins.

Borna disease virus is a unique neurotropic RNA virus that causes neurologic disease in a wide variety of animal hosts. We established an enzyme-linked immunosorbent assay for the detection of antibodies to Borna disease virus on the basis of the use of three recombinant viral proteins (recp40, recp23, and recp18). This assay system is more sensitive and rapid than the methods currently used for the serologic diagnosis of infection such as Western blotting (immunoblotting), indirect immunofluorescence test, or immunoprecipitation.

Borna disease virus is a neurotropic negative-strand RNA virus that infects a wide range of vertebrate hosts, causing disturbances in movement and behavior. We have cloned and sequenced the 8910-nucleotide viral genome by using RNA from Borna disease virus particles. The viral genome has complementary 3' and 5' termini and contains antisense information for five open reading frames. Homology to Filoviridae, Paramyxoviridae, and Rhabdoviridae is found in both cistronic and extracistronic regions. Northern analysis indicates that the virus transcribes mono- and polycistronic RNAs and uses termination/polyadenylylation signals reminiscent of those observed in other negative-strand RNA viruses. Borna disease virus is likely to represent a previously unrecognized genus, bornaviruses, or family, Bornaviridae, within the order Mononegavirales.

Borna disease (BD) is a neurologic syndrome characterized by behavioral disturbances and the accumulation of specific proteins in limbic system neurons. A viral etiology has been proposed because BD can be induced in birds, rodents, and primates by inoculation with filtered brain homogenates from animals with BD. We report here the isolation and preliminary characterization of cDNA clones from a rat with BD. These clones hybridized to specific transcripts in BD rat brain and arrested in vitro translation of BD proteins. In situ hybridization experiments using RNA probes prepared from these clones showed an abundance of these transcripts in limbic system neurons. Northern (RNA) hybridizations using these RNA probes indicated that the BD agent is probably a virus with major transcripts of 8.5, 2.1, and 0.8 kilobases.

ABSTRACTThe 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. hesperidusbat 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.IMPORTANCEGlobal 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.

ABSTRACTDescribing 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.IMPORTANCEHepatitis 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.

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.

ABSTRACTFrom 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.IMPORTANCEThe 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.