Melioidosis in a Resident of Texas with No Recent Travel History, United States.

To our knowledge, environmental isolation of Burkholderia pseudomallei, the causative agent of melioidosis, from the continental United States has not been reported. We report a case of melioidosis in a Texas resident. Genomic analysis indicated that the isolate groups with B. pseudomallei isolates from patients in the same region, suggesting possible endemicity to this region.

First Reported Human Cases of Leptospirosis in the United States Virgin Islands in the Aftermath of Hurricanes Irma and Maria, September-November 2017.

OBJECTIVE: Following Hurricanes Irma and Maria, the first case of human leptospirosis ever identified in the US Virgin Islands (USVI) was reported to the Virgin Islands Department of Health. Leptospirosis is a potentially fatal bacterial disease caused by Leptospira species found in animal urine and urine-contaminated water and soil. Outbreaks can occur following extreme weather events. METHOD: Additional cases of leptospirosis were identified in the 2.5 months post-hurricanes by reviewing emergency department (ED) records from territorial hospitals for patients demonstrating leptospirosis-consistent symptoms, testing symptomatic patients previously enrolled in the USVI arbovirus surveillance system (VIASS), and adding leptospirosis testing prospectively to VIASS. Available patient sera underwent local rapid diagnostic testing for anti-Leptospira IgM followed by confirmatory microscopic agglutination testing at the US Centers for Disease Control and Prevention. Water was collected from cisterns with epidemiologic links to confirmed cases and tested by real-time PCR (qPCR) for pathogenic Leptospira spp. RESULTS: Sixteen retrospectively identified symptomatic patients were enrolled in VIASS; 15 with available samples tested negative. Based on review of 5226 ED charts, 6 patients were further investigated; of these, 5 were tested of which 1 was positive. Prospective leptospirosis surveillance tested 57 additional patients; of these, 1 was positive. Water from 1 of 5 tested cisterns was found positive by qPCR. CONCLUSIONS: This investigation documents the first 3 cases of leptospirosis reported in the USVI and demonstrates how VIASS successfully was adapted to establish leptospirosis surveillance. Contaminated cistern water was identified as a potential source for Leptospira spp. transmission, highlighting the need for additional post-hurricane remediation and disinfection guidance.

Comparison of nucleic acid extraction methods for next-generation sequencing of avian influenza A virus from ferret respiratory samples.

Influenza A virus is a negative-sense RNA virus with a segmented genome consisting of eight RNA segments. Avian influenza A virus (AIV) primarily infects avian hosts and sporadically infects mammals, which can lead to adaptation to new species. Next-generation sequencing (NGS) of emerging AIV genomes extracted from respiratory samples collected on sequential days from animal models and clinical patients enables analysis of the emergence of evolutionary variants within the virus population over time. However, obtaining codon complete AIV genome at a sufficient coverage depth for nucleotide variant calling remains a challenge, especially from post-inoculation respiratory samples collected at late time points that have low viral titers. In this study, nasal wash samples from ferrets inoculated with different subtypes of AIV were collected on various days post-inoculation. Each nasal wash sample was aliquoted and extracted using five commercially available nucleic acid extraction methods. Extracted influenza virus RNA was amplified and NGS conducted using Illumina Mi-Seq. For each nasal wash sample, completeness of AIV genome segments and coverage depth were compared among five extraction methods. Nucleic acids extracted by MagNA pure compact RNA isolation consistently yielded codon complete sequences for all eight genome segments at the required coverage depth at each time point sampled. The study revealed that DNase treatment was critical to the amplification of influenza genome segments and the downstream success of codon complete NGS from nasal wash samples. The findings from this study can be applied to improve NGS of influenza and other RNA viruses that infect the respiratory tract and are collected from respiratory samples.

Avian Influenza A(H7N2) Virus in Human Exposed to Sick Cats, New York, USA, 2016.

An outbreak of influenza A(H7N2) virus in cats in a shelter in New York, NY, USA, resulted in zoonotic transmission. Virus isolated from the infected human was closely related to virus isolated from a cat; both were related to low pathogenicity avian influenza A(H7N2) viruses detected in the United States during the early 2000s.

Molecular basis of mammalian transmissibility of avian H1N1 influenza viruses and their pandemic potential.

North American wild birds are an important reservoir of influenza A viruses, yet the potential of viruses in this reservoir to transmit and cause disease in mammals is not well understood. Our surveillance of avian influenza viruses (AIVs) at Delaware Bay, USA, revealed a group of similar H1N1 AIVs isolated in 2009, some of which were airborne-transmissible in the ferret model without prior adaptation. Comparison of the genomes of these viruses revealed genetic markers of airborne transmissibility in the Polymerase Basic 2 (PB2), PB1, PB1-F2, Polymerase Acidic-X (PA-X), Nonstructural Protein 1 (NS1), and Nuclear Export Protein (NEP) genes. We studied the role of NS1 in airborne transmission and found that NS1 mutants that were not airborne-transmissible caused limited tissue pathology in the upper respiratory tract (URT). Viral maturation was also delayed, evident as strong intranuclear staining and little virus at the mucosa. Our study of this naturally occurring constellation of genetic markers has provided insights into the poorly understood phenomenon of AIV airborne transmissibility by revealing a role for NS1 and characteristics of viral replication in the URT that were associated with airborne transmission. The transmissibility of these viruses further highlights the pandemic potential of AIVs in the wild bird reservoir and the need to maintain surveillance.

Role of domestic ducks in the emergence of a new genotype of highly pathogenic H5N1 avian influenza A viruses in Bangladesh.

Highly pathogenic avian influenza H5N1 viruses were first isolated in Bangladesh in February 2007. Subsequently, clades 2.2.2, 2.3.4.2 and 2.3.2.1a were identified in Bangladesh, and our previous surveillance data revealed that by the end of 2014, the circulating viruses exclusively comprised clade 2.3.2.1a. We recently determined the status of circulating avian influenza viruses in Bangladesh by conducting surveillance of live poultry markets and waterfowl in wetland areas from February 2015 through February 2016. Until April 2015, clade 2.3.2.1a persisted without any change in genotype. However, in June 2015, we identified a new genotype of H5N1 viruses, clade 2.3.2.1a, which quickly became predominant. These newly emerged H5N1 viruses contained the hemagglutinin, neuraminidase and matrix genes of circulating 2.3.2.1a Bangladeshi H5N1 viruses and five other genes of low pathogenic Eurasian-lineage avian influenza A viruses. Some of these internal genes were closely related to those of low pathogenic viruses isolated from ducks in free-range farms and wild birds in a wetland region of northeastern Bangladesh, where commercially raised domestic ducks have frequent contact with migratory birds. These findings indicate that migratory birds of the Central Asian flyway and domestic ducks in the free-range farms in Tanguar haor-like wetlands played an important role in the emergence of this novel genotype of highly pathogenic H5N1 viruses.

H1N1 influenza viruses varying widely in hemagglutinin stability transmit efficiently from swine to swine and to ferrets.

A pandemic-capable influenza virus requires a hemagglutinin (HA) surface glycoprotein that is immunologically unseen by most people and is capable of supporting replication and transmission in humans. HA stabilization has been linked to 2009 pH1N1 pandemic potential in humans and H5N1 airborne transmissibility in the ferret model. Swine have served as an intermediate host for zoonotic influenza viruses, yet the evolutionary pressure exerted by this host on HA stability was unknown. For over 70 contemporary swine H1 and H3 isolates, we measured HA activation pH to range from pH 5.1 to 5.9 for H1 viruses and pH 5.3 to 5.8 for H3 viruses. Thus, contemporary swine isolates vary widely in HA stability, having values favored by both avian (pH >5.5) and human and ferret (pH ≤5.5) species. Using an early 2009 pandemic H1N1 (pH1N1) virus backbone, we generated three viruses differing by one HA residue that only altered HA stability: WT (pH 5.5), HA1-Y17H (pH 6.0), and HA2-R106K (pH 5.3). All three replicated in pigs and transmitted from pig-to-pig and pig-to-ferret. WT and R106 viruses maintained HA genotype and phenotype after transmission. Y17H (pH 6.0) acquired HA mutations that stabilized the HA protein to pH 5.8 after transmission to pigs and 5.5 after transmission to ferrets. Overall, we found swine support a broad range of HA activation pH for contact transmission and many recent swine H1N1 and H3N2 isolates have stabilized (human-like) HA proteins. This constitutes a heightened pandemic risk and underscores the importance of ongoing surveillance and control efforts for swine viruses.

Highly Pathogenic Reassortant Avian Influenza A(H5N1) Virus Clade 2.3.2.1a in Poultry, Bhutan.

Highly pathogenic avian influenza A(H5N1), clade 2.3.2.1a, with an H9-like polymerase basic protein 1 gene, isolated in Bhutan in 2012, replicated faster in vitro than its H5N1 parental genotype and was transmitted more efficiently in a chicken model. These properties likely help limit/eradicate outbreaks, combined with strict control measures.

The Continuing Evolution of H5N1 and H9N2 Influenza Viruses in Bangladesh Between 2013 and 2014.

In 2011, avian influenza surveillance at the Bangladesh live bird markets (LBMs) showed complete replacement of the highly pathogenic avian influenza (HPAI) H5N1 virus of clade 2.2.2 (Qinghai-like H5N1 lineage) by the HPAI H5N1 clade 2.3.2.1. This clade, which continues to circulate in Bangladesh and neighboring countries, is an intra-and interclade reassortant; its HA, polymerase basic 1 (PB1), polymerase (PA), and nonstructural (NS) genes come from subclade 2.3.2.1a; the polymerase basic 2 (PB2) comes from subclade 2.3.2.1c; and the NA, nucleocapsid protein (NP), and matrix (M) gene from clade 2.3.4.2. The H9N2 influenza viruses cocirculating in the Bangladesh LBMs are also reassortants, possessing five genes (NS, M, NP, PA, and PB1) from an HPAI H7N3 virus previously isolated in Pakistan. Despite frequent coinfection of chickens and ducks, reassortment between these H5N1 and H9N2 viruses has been rare. However, all such reassortants detected in 2011 through 2013 have carried seven genes from the local HPAI H5N1 lineage and the PB1 gene from the Bangladeshi H9N2 clade G1 Mideast, itself derived from HPAI H7N3 virus. Although the live birds we sampled in Bangladesh showed no clinical signs of morbidity, the emergence of this reassortant HPAI H5N1 lineage further complicates endemic circulation of H5N1 viruses in Bangladesh, posing a threat to both poultry and humans.

Influenza surveillance on 'foie gras' duck farms in Bulgaria, 2008-2012.

OBJECTIVES: Ducks can shed and spread influenza A viruses (IAVs) while showing no disease signs. Our objective was to clarify the role of 'foie gras' ducks in the circulation of IAVs in Bulgaria. METHODS: Monthly avian influenza surveillance was conducted on 63 'foie gras' duck farms, 52 of which were surveyed throughout the study between November 2008 and April 2012. Virologic and serologic samples were collected and tested. During this time, wild bird samples were collected at major wild bird-resting areas near the Black Sea coast and Danube River. RESULTS: The study showed high isolation frequency of low-pathogenicity avian influenza viruses. In the raising population (<75 days old), subtypes H3, H4, and H6 were detected monthly and H5 LPAIV, sporadically. Different subtypes (H1, H10, H11) were isolated from the fattening premises (75- to 100-day-old ducks), suggesting different routes of introduction. Only 6 of the 52 farms that were surveyed both virologically and serologically were influenza-free throughout the study, possibly due to higher biosecurity measures implemented. No evidence of direct transmission of IAV from wild birds was found. Wild bird surveillance showed low isolation frequency of IAV. IAV prevalence of 0·55% for migratory ducks and 0·53% for migratory geese was estimated in November-December 2011 and January-February 2012, respectively, at two ornithologically important locations near the Black Sea coast. CONCLUSIONS: The 'foie gras' duck farms in Bulgaria are an optimal niche where Eurasian-like IAVs are maintained and reassorted unapparent to farmers and veterinarians.

Multiple introductions of highly pathogenic avian influenza H5N1 viruses into Bangladesh.

Highly pathogenic H5N1 and low pathogenic H9N2 influenza viruses are endemic to poultry markets in Bangladesh and have cocirculated since 2008. H9N2 influenza viruses circulated constantly in the poultry markets, whereas highly pathogenic H5N1 viruses occurred sporadically, with peaks of activity in cooler months. Thirty highly pathogenic H5N1 influenza viruses isolated from poultry were characterized by antigenic, molecular, and phylogenetic analyses. Highly pathogenic H5N1 influenza viruses from clades 2.2.2 and 2.3.2.1 were isolated from live bird markets only. Phylogenetic analysis of the 30 H5N1 isolates revealed multiple introductions of H5N1 influenza viruses in Bangladesh. There was no reassortment between the local H9N2 influenza viruses and H5N1 genotype, despite their prolonged cocirculation. However, we detected two reassortant H5N1 viruses, carrying the M gene from the Chinese H9N2 lineage, which briefly circulated in the Bangladesh poultry markets and then disappeared. On the other hand, interclade reassortment occurred within H5N1 lineages and played a role in the genesis of the currently dominant H5N1 viruses in Bangladesh. Few 'human-like' mutations in H5N1 may account for the limited number of human cases. Antigenically, clade 2.3.2.1 H5N1 viruses in Bangladesh have evolved since their introduction and are currently mainly homogenous, and show evidence of recent antigenic drift. Although reassortants containing H9N2 genes were detected in live poultry markets in Bangladesh, these reassortants failed to supplant the dominant H5N1 lineage.

Spread of influenza virus A (H5N1) clade 2.3.2.1 to Bulgaria in common buzzards.

On March 15, 2010, a highly pathogenic avian influenza virus was isolated from the carcass of a common buzzard (Buteo buteo) in Bulgaria. Phylogenetic analyses of the virus showed a close genetic relationship with influenza virus A (H5N1) clade 2.3.2.1 viruses isolated from wild birds in the Tyva Republic and Mongolia during 2009-2010. Designated A/common buzzard/Bulgaria/38WB/2010, this strain was highly pathogenic in chickens but had low pathogenicity in mice and ferrets and no molecular markers of increased pathogenicity in mammals. The establishment of clade 2.3.2.1 highly pathogenic avian influenza viruses of the H5N1 subtype in wild birds in Europe would increase the likelihood of health threats to humans and poultry in the region.