Detection and Characterization of Swine Origin Influenza A(H1N1) Pandemic 2009 Viruses in Humans following Zoonotic Transmission.

Human-to-swine transmission of seasonal influenza viruses has led to sustained human-like influenza viruses circulating in the U.S. swine population. While some reverse zoonotic-origin viruses adapt and become enzootic in swine, nascent reverse zoonoses may result in virus detections that are difficult to classify as "swine-origin" or "human-origin" due to the genetic similarity of circulating viruses. This is the case for human-origin influenza A(H1N1) pandemic 2009 (pdm09) viruses detected in pigs following numerous reverse zoonosis events since the 2009 pandemic. We report the identification of two human infections with A(H1N1)pdm09 viruses originating from swine hosts and classify them as "swine-origin" variant influenza viruses based on phylogenetic analysis and sequence comparison methods. Phylogenetic analyses of viral genomes from two cases revealed these viruses were reassortants containing A(H1N1)pdm09 hemagglutinin (HA) and neuraminidase (NA) genes with genetic combinations derived from the triple reassortant internal gene cassette. Follow-up investigations determined that one individual had direct exposure to swine in the week preceding illness onset, while another did not report swine exposure. The swine-origin A(H1N1) variant cases were resolved by full genome sequence comparison of the variant viruses to swine influenza genomes. However, if reassortment does not result in the acquisition of swine-associated genes and swine virus genomic sequences are not available from the exposure source, future cases may not be discernible. We have developed a pipeline that performs maximum likelihood analyses, a k-mer-based set difference algorithm, and random forest algorithms to identify swine-associated sequences in the hemagglutinin gene to differentiate between human-origin and swine-origin A(H1N1)pdm09 viruses.IMPORTANCE Influenza virus infects a wide range of hosts, resulting in illnesses that vary from asymptomatic cases to severe pneumonia and death. Viral transfer can occur between human and nonhuman hosts, resulting in human and nonhuman origin viruses circulating in novel hosts. In this work, we have identified the first case of a swine-origin influenza A(H1N1)pdm09 virus resulting in a human infection. This shows that these viruses not only circulate in swine hosts, but are continuing to evolve and distinguish themselves from previously circulating human-origin influenza viruses. The development of techniques for distinguishing human-origin and swine-origin viruses are necessary for the continued surveillance of influenza viruses. We show that unique genetic signatures can differentiate circulating swine-associated strains from circulating human-associated strains of influenza A(H1N1)pdm09, and these signatures can be used to enhance surveillance of swine-origin influenza.

Genetic diversity and evolution of the emerging picornavirus Senecavirus A.

Senecavirus A (SVA) is an emerging picornavirus that causes vesicular disease (VD) in swine. The virus has been circulating in swine in the United Stated (USA) since at least 1988, however, since 2014 a marked increase in the number of SVA outbreaks has been observed in swine worldwide. The factors that led to the emergence of SVA remain unknown. Evolutionary changes that accumulated in the SVA genome over the years may have contributed to the recent increase in disease incidence. Here we compared full-genome sequences of historical SVA strains (identified before 2010) from the USA and global contemporary SVA strains (identified after 2011). The results from the genetic analysis revealed 6.32 % genetic divergence between historical and contemporary SVA isolates. Selection pressure analysis revealed that the SVA polyprotein is undergoing selection, with four amino acid (aa) residues located in the VP1 (aa 735), 2A (aa 941), 3C (aa 1547) and 3D (aa 1850) coding regions being under positive/diversifying selection. Several aa substitutions were observed in the structural proteins (VP1, VP2 and VP3) of contemporary SVA isolates when compared to historical SVA strains. Some of these aa substitutions led to changes in the surface electrostatic potential of the structural proteins. This work provides important insights into the molecular evolution and epidemiology of SVA.

Influenza A(H3N2) Virus in Swine at Agricultural Fairs and Transmission to Humans, Michigan and Ohio, USA, 2016.

In 2016, a total of 18 human infections with influenza A(H3N2) virus occurred after exposure to influenza-infected swine at 7 agricultural fairs. Sixteen of these cases were the result of infection by a reassorted virus with increasing prevalence among US swine containing a hemagglutinin gene from 2010-11 human seasonal H3N2 strains.

Outbreak of Influenza A(H3N2) Variant Virus Infections Among Persons Attending Agricultural Fairs Housing Infected Swine - Michigan and Ohio, July-August 2016.

On August 3, 2016, the Ohio Department of Health Laboratory reported to CDC that a respiratory specimen collected on July 28 from a male aged 13 years who attended an agricultural fair in Ohio during July 22-29, 2016, and subsequently developed a respiratory illness, tested positive by real-time reverse transcription-polymerase chain reaction (rRT-PCR) for influenza A(H3N2) variant* (H3N2v). The respiratory specimen was collected as part of routine influenza surveillance activities. The next day, CDC was notified of a child aged 9 years who was a swine exhibitor at an agricultural fair in Michigan who became ill on July 29, 2016, and tested positive for H3N2v virus at the Michigan Department of Health and Human Services Laboratory. Investigations by Michigan and Ohio health authorities identified 18 human infections linked to swine exhibits at agricultural fairs. To minimize transmission of influenza viruses from infected swine to visitors, agricultural fair organizers should consider prevention measures such as shortening the time swine are on the fairgrounds, isolating ill swine, maintaining a veterinarian on call, providing handwashing stations, and prohibiting food and beverages in animal barns. Persons at high risk for influenza-associated complications should be discouraged from entering swine barns.

Vesicular stomatitis outbreak in the southwestern United States, 2012.

Vesicular stomatitis is a viral disease primarily affecting horses and cattle when it occurs in the United States. Outbreaks in the southwestern United States occur sporadically, with initial cases typically occurring in Texas, New Mexico, or Arizona and subsequent cases occurring in a northward progression. The viruses causing vesicular stomatitis can be transmitted by direct contact of lesioned animals with other susceptible animals, but transmission is primarily through arthropod vectors. In 2012, an outbreak of vesicular stomatitis in the United States occurred that was caused by Vesicular stomatitis New Jersey virus serotype. Overall, 51 horses on 36 premises in 2 states were confirmed positive. Phylogenetic analysis of the virus indicated that it was most closely related to viruses detected in the state of Veracruz, Mexico, in 2000.

Identification of the benign mesenchymal tumor gene HMGA2 in lymphangiomyomatosis.

The normal expression pattern of HMGA2, an architectural transcription factor, is primarily restricted to cells of the developing mesenchyme before their overt differentiation during organogenesis. A detailed in situ hybridization analysis showed that the undifferentiated mesoderm of the embryonic lung expressed Hmga2 but it was not expressed in the newborn or adult lung. Previously, HMGA2 was shown to be misexpressed in a number of benign, differentiated mesenchymal tumors including lipomas, uterine leiomyomas, and pulmonary chondroid hamartomas. Here, we show that HMGA2 is misexpressed in pulmonary lymphangiomyomatosis (LAM), a severe disorder of unknown etiology consisting of lymphatic smooth muscle cell proliferation that results in the obstruction of airways, lymphatics, and vessels. Immunohistochemistry was done with antibodies to HMGA2 and revealed expression in lung tissue samples obtained from 21 patients with LAM. In contrast, HMGA2 was not expressed in sections of normal adult lung or other proliferative interstitial lung diseases, indicating that the expression of HMGA2 in LAM represents aberrant gene activation and is not due solely to an increase in cellular proliferation. In vivo studies in transgenic mice show that misexpression of HMGA2 in smooth muscle cells resulted in increased proliferation of these cells in the lung surrounding the epithelial cells. Therefore, similar to the other mesenchymal neoplasms, HMGA2 misexpression in the smooth muscle cell leads to abnormal proliferation and LAM tumorigenesis. These results suggest that HMGA2 plays a central role in the pathogenesis of LAM and is a potential candidate as a therapeutic target.

Neuroleptic malignant syndrome and serotonin syndrome in the critical care setting: case analysis.

BACKGROUND: Serotonin syndrome (SS) and neuroleptic malignant syndrome (NMS) are medical emergencies associated with psychotropic administration. Differentiation and treatment can be complex, especially when features of both syndromes are present and the patient has taken both serotonergic and neuroleptic agents. METHOD: Case analysis of a poly-drug overdose (venlafaxine, topiramate, divalproex sodium, risperidone, and carbamazepine) presenting with mixed SS/NMS features and whose clinical management suggests a practical algorithm for treatment of undifferentiated SS/NMS in critical care settings. RESULTS: The suggested algorithm includes: 1) Supportive care and withdrawal of all potentially offending agents; 2) Laboratory evaluation with prompt initiation of treatment for both disorders--cyproheptadine for SS and dantrolene for NMS; 3) Do not use bromocriptine (contraindicated in SS) or chlorpromazine (contraindicated in NMS) initially; 4) Add bromocriptine when clinical presentation becomes consistent with NMS (SS can be prolonged if serotonergic agent has long half-life). CONCLUSIONS: Prompt and appropriate identification and intervention are essential for successful management of SS and NMS. The suggested treatment algorithm allows for specific treatment of both disorders and avoids potentially exacerbating either one. The algorithm derived from this case could serve as both a practical guideline and impetus for further investigation in light of increasing psychotropic co-administration.

Hmga2 promoter analysis in transgenic mice.

HMGA2(2) belongs to the high mobility group A (HMGA) family of architectural transcription factors which participate in a wide variety of nuclear processes ranging from transcription to recombination, playing an important role in chromatin remodelling. HMGA2 is expressed during embryogenesis but not by adult somatic tissues, yet it becomes re-expressed following neoplastic transformation. A role in development is underscored by the finding that the inactivation of the Hmga2 gene is responsible for the murine pygmy phenotype. To elucidate mechanisms that control HMGA2 expression, we have previously cloned the gene and identified functional elements involved in its regulation. In this paper, transgenic mice were generated to define genomic regions involved in Hmga2 developmental and tissue-specific transcriptional regulation. A genomic region from -8.1 to -3.7kb upstream from the initiation site has been found to recapitulate most of the spatial and temporal endogenous Hmga2 gene expression.

Hmga1 is required for normal sperm development.

The Hmgi protein family of chromosomal architectural factors is extensively studied for its roles in embryogenesis and its association with benign mesenchymal tumors. Although the biochemical function of Hmga1 has been studied in vitro, to provide in vivo insight into its biological function, a targeted disruption of Hmga1 was initiated. Chimeric founder mice were derived from embryonic stem (ES) cells harboring a targeted mutation in a single Hmga1 allele. These 14 different chimeric founders produced 494 black progeny. Since none of these 494 progeny were agouti, none of them were derived from ES cells. Control injections of the wild-type ES cell lines resulted in ES cell derived agouti mice, indicating that the ES cells were totipotent. Therefore, our results indicate that one intact Hmga1 allele was not sufficient for germ-line transmission of the ES cells. Seven chimeric founder mice that were examined histologically demonstrated aberrant regions in their reproductive organs. Aberrant regions of seminiferous tubules were reduced in diameter, demonstrated vacuolated Sertoli cells, and had an absolute deficiency of sperm. While the Hmga1(+/-) ES cells were shown to contribute to the formation of the epididymides, they did not significantly contribute to the testes of chimeric founder mice. No sperm isolated from any of the Hmga1(+/-) chimeric mice were shown to arise from the ES cells, as none of them contained the targeted disruption of the Hmga1 gene. Our results suggest that both alleles of Hmga1 are required for normal sperm production in the mouse.

Emphysematous changes are caused by degradation of type III collagen in transgenic mice expressing MMP-1.

Disruption of the extracellular matrix is believed to play an important role in the pathogenesis of emphysema. Prior studies have demonstrated that transgenic mice expressing the human tissue collagenase, matrix metalloproteinase 1 (MMP-1), develop emphysema. MMP-1 is a protease with substrate specificity for fibrillar collagen. Type I and III collagens, which are the most abundant proteins within the lungs, are the primary substrates for MMP-1. To assess if type I collagen was indeed the site of action for MMP-1 in these transgenic mice, hybrid mice were generated by crossing the MMP-1 transgenic mice with mice that had degradation-resistant type I collagen. The hybrid mice demonstrated an identical emphysematous phenotype as the MMP-1 transgenic mice, indicating that the degradation of type I collagen was not essential to the development of emphysema in these mice. Immunohistochemical studies in control mice demonstrated that collagen fibers in the alveolar walls and ducts of the normal mouse lungs consist mainly of type III collagen. In the transgenic and hybrid mice, the emphysematous changes, which developed, were associated with a marked decrease in type III collagen in these alveolar structures. These results indicate that MMP-1 generated the emphysematous phenotype via the degradative effect on type III collagen, which is a vital structural element of the alveolar walls. This is the first study to show that a matrix metalloproteinase may cause emphysema via its effects on a specific collagen subtype. As such, it should provide important insight into the mechanisms of this disease in humans.

Hmgi-c-independent Activation of MuRantes in Vivo.

The architectural factor HMGI-C is of considerable interest for its recognized roles in mammalian development and tumorigenesis. As a result, the identification of downstream target genes of HMGI-C is the present focus of active research. In vitro evidence from macrophage cell lines has previously suggested that Hmgi-c is necessary for the inducible activation of MuRantes expression. To attempt to verify this hypothesis, an in vivo analysis was performed that took advantage of the existence of the Hmgi-c null mouse strain. The ability of cells and tissues extracted from Hmgi-c null mice to express the inflammatory chemokine MuRantes was investigated. The investigation examined MuRantes expression in primary embryonic fibroblasts and fresh peritoneal macrophages after Newcastle disease virus induction and whole organs after lipopolysaccharide induction. Each of these systems clearly demonstrates that Hmgi-c is not required for the activation of MuRantes expression.