System Infections In Humans Research Articles

Developing nanosize carrier systems for Amphotericin-B: A review on the biomedical application of nanoparticles for the treatment of leishmaniasis and fungal infections.

New formulations of Amphotericin-B (Am-B), the most popular therapeutic drug for many human infections such as parasitic and fungal pathogens, are safe, economical, and effective in the world. Several newly designed carrier systems for Am-B can also be considered orally with sufficient gastrointestinal permeability and good solubility. However, the clinical application of several new formulations of Am-B with organ cytotoxicity, low bioavailability, high costs, and technical problems have caused some issues. Therefore, more attention and scientific design are required to progress safe and effective drug delivery systems. Currently, the application of nano-based technology and nanomaterials in the advancement of drug delivery systems exhibits promising outcomes to cure many human systemic infections. Designing novel drug delivery systems including solid lipid nanostructured materials, lipo-polymersomes, drug conjugates and microneedles, liposomes, polymer and protein-based nanostructured materials, dendrimers, emulsions, mixed micelles, polymeric micelles, cyclodextrins, nanocapsules, and nanocochleate for Am-B has many advantages to reducing several related issues. The unique properties of nanostructured particles such as proper morphology, small size, surface coatings, and, electrical charge, permit scientists to design new nanocomposite materials against microorganisms for application in various human diseases. These features have made these nanoparticles an ideal candidate for drug delivery systems in clinical approaches to cure a number of human disorders and currently, several therapeutic nanostructured material formulations are under different stages of clinical tests. Hence, this scientific paper mainly discussed the advances in new formulations of Am-B for the treatment of human systemic infections and related clinical tests.

The origin of human pathogenicity and biological interactions in Chaetothyriales

Fungi in the order Chaetothyriales are renowned for their ability to cause human infections. Nevertheless, they are not regarded as primary pathogens, but rather as opportunists with a natural habitat in the environment. Extremotolerance is a major trend in the order, but quite different from black yeasts in Capnodiales which focus on endurance, an important additional parameter is advancing toxin management. In the ancestral ecology of rock colonization, the association with metabolite-producing lichens is significant. Ant-association, dealing with pheromones and repellents, is another mainstay in the order. The phylogenetically derived family, Herpotrichiellaceae, shows dual ecology in monoaromatic hydrocarbon assimilation and the ability to cause disease in humans and cold-blooded vertebrates. In this study, data on ecology, phylogeny, and genomics were collected and analyzed in order to support this hypothesis on the evolutionary route of the species of Chaetothyriales. Comparing the ribosomal tree with that of enzymes involved in toluene degradation, a significant expansion of cytochromes is observed and the toluene catabolism is found to be complete in some of the Herpotrichiellaceae. This might enhance human systemic infection. However, since most species have to be traumatically inoculated in order to cause disease, their invasive potential is categorized as opportunism. Only in chromoblastomycosis, true pathogenicity might be surmised. The criterion would be the possible escape of agents of vertebrate disease from the host, enabling dispersal of adapted genotypes to subsequent generations.

Talaromyces marneffei Can Capture CD86 Proteins of Macrophages in vitro

Talaromyces marneffei (T. marneffei) is a thermally dimorphic fungus endemic to Southeast Asia that causes human systemic infection. Our earlier immunohistochemical studies revealed that the organisms were markedly labeled with the CD86 antigen in cutaneous lesions brought on by infection. However, the relationship between T. marneffei and the CD86 co-stimulatory molecule is still unknown. To explore the association between CD86 Protein and Talaromyces marneffei organisms in vitro and discuss the potential mechanisms. We created the CD86-EGFP fusion protein in THP-1 macrophages and co-cultured T. marneffei conidia with it. We used confocal fluorescence microscopy to view in vitro dynamics. The link between CD86 Protein and Talaromyces marneffei organisms in vitro was discovered using immunoelectron microscopy, indirect immunofluorescence test, and immunohistochemistry assay. T. marneffei cells received soluble CD86-EGFP from THP-1 macrophages detected by confocal fluorescent microscopy. Both the indirect immunofluorescence assay and the immunohistochemical assay showed that T. marneffei conidia were stained by the CD86 marker. Immunoelectron microscopy showed that characteristic colloidal gold particles were observed in T. marneffei organisms when co-cultured with THP-1 macrophages. T. marneffei organisms have the ability to capture CD86 proteins from macrophages in vitro.

Alt solunum yolu enfeksiyonu olan köpeklerde canine coronavirusun tespiti ve moleküler karakterizasyonu

Coronaviridae familyasında yer alan coronaviruslar insan ve hayvanlarda sindirim ve solunum sistemi enfeksiyonlarına neden olmaktadır. Alfacoronavirus içerisinde yer alan canine coronaviruslar (CCoV)’ın CCoV I ve CCoV II olmak üzere iki alt tipi bulunmaktadır. CCoV-II ise CCoV- IIa ve IIb olmak üzere iki genotipe ayrılmaktadır. CCoV her yaştaki ve her türlü beslenme şekline sahip köpekleri etkilemesine rağmen özellikle yeni doğan yavrular daha duyarlı ve ciddi şekilde etkilenebilmektedir. Yapılan literatür araştırmalarına göre ülkemizde özellikle alt solunum yolu enfeksiyonlarındaki canine coronavirus varlığının tespitine yönelik moleküler çalışmalara rastlanılmamıştır. Bu çalışmada alt solunum yolu enfeksiyonu tanımlanan barınak köpeklerinde CCoV’un tespiti ve moleküler karakterizasyonunun yapılması amaçlandı. Bu amaçla alt solunum yolu enfeksiyonu tespit edilen 40 adet barınak köpeğinden alınan Bronkoalveolar Lavaj (BAL) sıvıları incelendi. Test edilen 40 köpeğe ait BAL sıvılarından 3 tanesinde CCoV tespit edildi. Yapılan dizin analizi sonrasında elde edilen dizinler ile filogenetik ağaç yapıldı. Filogenetik ağaçta pozitif bulunan 3 örnekten 2 sinin CCoV-I, bir örneğin ise CCoV-II olduğu tespit edildi. Sonuç olarak bu çalışma ile barınak köpeklerinin alt solunum yolu rahatsızlıklarında CCoV-I ve CCoV-II’ nin rol oynayabileceği ortaya konulmuştur. Ayrıca aynı barınakta farklı hayvanlarda iki farklı CCoV’nun tespiti önemli bir veri olarak değerlendirilmiş olup özellikle barınak koşulları gibi kalabalık ortamda barındırılan köpeklerdeki her iki tipin tespiti gelecekte oluşabilecek yeni varyantların ya da alt tiplerin oluşabilme ihtimalinin de göz ardı edilmemesi gerektiğini göstermektedir.

Campylobacter fetus subspecies venerealis meningitis associated with a companion dog in a young adult: a case report

BackgroundCampylobacter spp., common commensals in the gastrointestinal tract of animals, especially poultry, can cause acute gastrointestinal illness in humans through animal-to-human transmission. Although Campylobacter fetus, especially subspecies fetus, rarely leads to systemic infections such as bacteremia in immunocompromised patients, it is unclear whether Campylobacter fetus subspecies venerealis (Cfv) causes infectious diseases in humans.Case presentationA 28-year-old man with a history of chronic alcoholism visited the emergency department with weakness of the left extremities. The patient was clinically diagnosed with community-acquired bacterial meningitis. The organism from the blood culture was subsequently identified as Campylobacter fetus. On phylogenetic analysis, the 16S rRNA sequence showed 99.93% similarity with other Cfv 16S rRNA sequences. The patient had no exposure to identifiable sources except for close contact with a companion dog, which could have been a possible source of transmission.ConclusionsThis case suggests that Cfv could lead to human systemic infections such as meningitis and that companion animals, in addition to well-known animal hosts, could be sources of transmission.

Further Characterization of Rio Grande Virus and Potential for Cross Reactivity with Rift Valley Fever Virus Assays.

Phleboviruses (genus Phlebovirus, family Phenuiviridae) are emerging pathogens of humans and animals. Sand-fly-transmitted phleboviruses are found in Europe, Africa, the Middle East, and the Americas, and are responsible for febrile illness and nervous system infections in humans. Rio Grande virus (RGV) is the only reported phlebovirus in the United States. Isolated in Texas from southern plains woodrats, RGV is not known to be pathogenic to humans or domestic animals, but serologic evidence suggests that sheep (Ovis aries) and horses (Equus caballus) in this region have been infected. Rift Valley fever virus (RVFV), a phlebovirus of Africa, is an important pathogen of wild and domestic ruminants, and can also infect humans with the potential to cause severe disease. The introduction of RVFV into North America could greatly impact U.S. livestock and human health, and the development of vaccines and countermeasures is a focus of both the CDC and USDA. We investigated the potential for serologic reagents used in RVFV diagnostic assays to also detect cells infected with RGV. Western blots and immunocytochemistry assays were used to compare the antibody detection of RGV, RVFV, and two other New World phlebovirus, Punta Toro virus (South and Central America) and Anhanga virus (Brazil). Antigenic cross-reactions were found using published RVFV diagnostic reagents. These findings will help to inform test interpretation to avoid false positive RVFV diagnoses that could lead to public health concerns and economically costly agriculture regulatory responses, including quarantine and trade restrictions.

Infección invasiva por Listeria monocytogenes en un paciente con síndrome de Evans.

Listeria monocytogenes es una bacteria intracelular facultativa que tiene predilección por causar infecciones del sistema nervioso central en humanos. Cuya población de riesgo predominante es lactantes, adultos mayores y en estados de inmunosupresión. Los principales síndromes clínicos causados por Listeria monocytogenes incluyen gastroenteritis febril, infección perinatal e infecciones sistémicas marcadas por infecciones del sistema nervioso central con o sin bacteriemia. Presentamos el caso de un paciente masculino de 66 años portador de síndrome de Evans en tratamiento inmunosupresor quien acude al servicio de emergencias del Hospital San Carlos el 27 de agosto de 2019, por un cuadro agudo de fiebre, cefalea y ataque al estado general en quien se documenta síndrome de respuesta inflamatoria sistémica y meningitis bacteriana ante lo cual inicio empíricamente cefotaxime. El panel molecular y los hemocultivos fueron positivos por Listeria monocytogenes. El paciente recibió 3 semanas de antibioticoterapia con ampicilina con excelente evolución clínica y sin secuelas neurológicas.

Identification and molecular typing of Naegleria fowleri from a patient with primary amebic meningoencephalitis in China

Naegleria fowleri is the only Naegleria spp. known to cause an acute, fulminant, and rapidly fatal central nervous system infection in humans called primary amebic meningoencephalitis (PAM). In 2016, a patient with suspected PAM was found in Zhejiang Province of China. The pathogen was identified by microscopic examination and PCR. The positive PCR products were sequenced and the sequences were aligned using the NCBI BLAST program. The homologous and phylogenetic analysis was conducted using MEGA 6 program. On microscopy of direct smears, motile cells with pseudopodia were observed, and the motion characteristics of the pseudopodia as well as the cell morphology suggested that the pathogens were amoeba trophozoites. Wright–Giemsa-stained smears showed amoeba trophozoites of various shapes, which measured 10–25μm in size; these were characterized by a prominent, centrally placed nucleolus and a vacuolated cytoplasm. PCR was negative for Entamoeba histolytica and Entamoeba dispar, but positive for Naegleria spp. and N. fowleri. The nucleotide sequences acquired in this study have been submitted to GenBank with accession numbers KX909928 and KX909927, respectively. The BLAST analysis revealed that the sequences of KX909928 and KX909927 had 100% similarity with the sequence of the N. fowleri gene (KT375442.1). Sequence alignment and the phylogenetic tree revealed that the N. fowleri collected in this study was classified as genotype 2 and was most closely related to Naegleria lovaniensis. This study confirmed N. fowleri as the agent responsible for the infection in this patient. PAM normally progresses rapidly and is generally universally fatal within a week. Unfortunately this patient died at 2 weeks after the onset of symptoms.

Genome sequencing of Chlamydia trachomatis serovars E and F reveals substantial genetic variation.

Chlamydia trachomatis (Ctr) is a bacterial pathogen that causes ocular, urogenital and lymph system infections in humans. It is highly abundant and among its serovars, E, F and D are most prevalent in sexually transmitted disease. However, the number of publicly available genome sequences of the serovars E and F, and thereby our knowledge about the molecular architecture of these serovars, is low. Here we sequenced the genomes of six E and F clinical isolates and one E lab strain, in order to study the genetic variance in these serovars. As observed before, the genomic variation inside the Ctr genomes is very low and the phylogenetic placement in comparison to publicly available genomes is as expected by ompA gene serotyping. However, we observed a large InDel carrying four to five open reading frames in one clinical E sample and in the E lab strain. We have also observed substantial variation on nucleotide and amino acid levels, especially in membrane proteins and secreted proteins. Furthermore, these two groups of proteins are also target for recombination events. One clinical F isolate was genetically heterogeneous and revealed the highest differences on nucleotide level in the pmpE gene.

A Selective Bottleneck Shapes the Evolutionary Mutant Spectra of Enterovirus A71 during Viral Dissemination in Humans.

RNA viruses accumulate mutations to rapidly adapt to environmental changes. Enterovirus A71 (EV-A71) causes various clinical manifestations with occasional severe neurological complications. However, the mechanism by which EV-A71 evolves within the human body is unclear. Utilizing deep sequencing and haplotype analyses of viruses from various tissues of an autopsy patient, we sought to define the evolutionary pathway by which enterovirus A71 evolves fitness for invading the central nervous system in humans. Broad mutant spectra with divergent mutations were observed at the initial infection sites in the respiratory and digestive systems. After viral invasion, we identified a haplotype switch and dominant haplotype, with glycine at VP1 residue 31 (VP1-31G) in viral particles disseminated into the integumentary and central nervous systems. In vitro viral growth and fitness analyses indicated that VP1-31G conferred growth and a fitness advantage in human neuronal cells, whereas VP1-31D conferred enhanced replication in human colorectal cells. A higher proportion of VP1-31G was also found among fatal cases, suggesting that it may facilitate central nervous system infection in humans. Our data provide the first glimpse of EV-A71 quasispecies from oral tissues to the central nervous system within humans, showing broad implications for the surveillance and pathogenesis of this reemerging viral pathogen.IMPORTANCE EV-A71 continues to be a worldwide burden to public health. Although EV-A71 is the major etiological agent of hand, foot, and mouth disease, it can also cause neurological pulmonary edema, encephalitis, and even death, especially in children. Understanding selection processes enabling dissemination and accurately estimating EV-A71 diversity during invasion in humans are critical for applications in viral pathogenesis and vaccine studies. Here, we define a selection bottleneck appearing in respiratory and digestive tissues. Glycine substitution at VP1 residue 31 helps viruses break through the bottleneck and invade the central nervous system. This substitution is also advantageous for replication in neuronal cells in vitro Considering that fatal cases contain enhanced glycine substitution at VP1-31, we suggest that the increased prevalence of VP1-31G may alter viral tropism and aid central nervous system invasion. Our findings provide new insights into a dynamic mutant spectral switch active during acute viral infection with emerging viral pathogens.

In vitro growth, cytopathic effects and clearance of monolayers by clinical isolates of Balamuthia mandrillaris in human skin cell cultures

Balamuthia mandrillaris is a free-living ameba (FLA) that has been isolated or its DNA identified in soil, dust and water. It causes a fatal central nervous system infection in humans and animals. Although it is environmental as Acanthamoeba and Naegleria fowleri, the two other free-living amebae that also cause CNS infections in humans and other animals, Balamuthia does not feed on bacteria as the other FLA. In the laboratory, it can be grown on a variety of mammalian cell cultures. In this study we examined the ability of three different Balamuthia isolates to grow on several different human skin cell cultures including the WT/A keratinocyte cell cultures. A corneal isolate of Acanthamoeba castellanii was used for comparison.

Novel Indole-2-Carboxamide Compounds Are Potent Broad-Spectrum Antivirals Active against Western Equine Encephalitis Virus In Vivo

Neurotropic alphaviruses, including western, eastern, and Venezuelan equine encephalitis viruses, cause serious and potentially fatal central nervous system infections in humans for which no currently approved therapies exist. We previously identified a series of thieno[3,2-b]pyrrole derivatives as novel inhibitors of neurotropic alphavirus replication, using a cell-based phenotypic assay (W. Peng et al., J. Infect. Dis. 199:950-957, 2009, doi:http://dx.doi.org/10.1086/597275), and subsequently developed second- and third-generation indole-2-carboxamide derivatives with improved potency, solubility, and metabolic stability (J. A. Sindac et al., J. Med. Chem. 55:3535-3545, 2012, doi:http://dx.doi.org/10.1021/jm300214e; J. A. Sindac et al., J. Med. Chem. 56:9222-9241, 2013, http://dx.doi.org/10.1021/jm401330r). In this report, we describe the antiviral activity of the most promising third-generation lead compound, CCG205432, and closely related analogs CCG206381 and CCG209023. These compounds have half-maximal inhibitory concentrations of ∼1 μM and selectivity indices of >100 in cell-based assays using western equine encephalitis virus replicons. Furthermore, CCG205432 retains similar potency against fully infectious virus in cultured human neuronal cells. These compounds show broad inhibitory activity against a range of RNA viruses in culture, including members of the Togaviridae, Bunyaviridae, Picornaviridae, and Paramyxoviridae families. Although their exact molecular target remains unknown, mechanism-of-action studies reveal that these novel indole-based compounds target a host factor that modulates cap-dependent translation. Finally, we demonstrate that both CCG205432 and CCG209023 dampen clinical disease severity and enhance survival of mice given a lethal western equine encephalitis virus challenge. These studies demonstrate that indole-2-carboxamide compounds are viable candidates for continued preclinical development as inhibitors of neurotropic alphaviruses and, potentially, of other RNA viruses. IMPORTANCE There are currently no approved drugs to treat infections with alphaviruses. We previously identified a novel series of compounds with activity against these potentially devastating pathogens (J. A. Sindac et al., J. Med. Chem. 55:3535-3545, 2012, doi:http://dx.doi.org/10.1021/jm300214e; W. Peng et al., J. Infect. Dis. 199:950-957, 2009, doi:http://dx.doi.org/10.1086/597275; J. A. Sindac et al., J. Med. Chem. 56:9222-9241, 2013, http://dx.doi.org/10.1021/jm401330r). We have now produced third-generation compounds with enhanced potency, and this manuscript provides detailed information on the antiviral activity of these advanced-generation compounds, including activity in an animal model. The results of this study represent a notable achievement in the continued development of this novel class of antiviral inhibitors.

Impact of diagnostic procedures on patient management and hospitalization cost during the 2000 and 2005 enterovirus epidemics in Marseilles, France

Enteroviruses are frequent aetiological agents of central nervous system infections in humans. In 2000 and 2005, two large outbreaks of Echovirus 30 (a member of species human enterovirus B) were observed in the University Hospitals of Marseilles (France). Between the two epidemics, the diagnostic protocols for enterovirus infection were modified, moving from viral cultures and classic RT-PCR in 2000 to real-time RT-PCR in 2005. We compared some viral and epidemiological characteristics of the 2000 and 2005 outbreaks with special attention to diagnostic procedures and to the subsequent clinical management of patients. Despite similar virological and epidemiological characteristics during both outbreaks, our results show that real-time RT-PCR techniques used in 2005 noticeably shortened the period of time necessary to deliver diagnostic results and suggest that this was associated with a decrease in the duration of hospitalization for -positive cases. In conclusion, this study suggests that the improvement of enterovirus diagnosis had a major financial impact on the management of the 2005 epidemic in Marseilles and may constitute an interesting example of how new diagnostic methods in microbiology can be self-financed through improvement in patient management.

Isolation, morphologic, serologic and molecular identification of Acanthamoeba T4 genotype from the liver of a Temminck's tragopan ( Tragopan temminckii)

Members of the genus Acanthamoeba are usually free-living amoebae that are found in a variety of ecological niches including soil, fresh and brackish water, dust in the air, heating, ventilating, and air conditioning filters, swimming pools and hot tubs. Occasionally they are also known to cause central nervous system infections in humans and animals. We isolated into culture an amoeba from the liver of a Temminck's tragopan (horned pheasant) ( Tragopan temminckii) that died of amoebic infection. We identified the infecting amoeba as Acanthamoeba sp. based on culture characteristics, cyst morphology and immunofluorescence assays. Additionally, we identified the amoeba as Acanthamoeba, genotype T4, by sequencing a diagnostic region of the nuclear small subunit ribosomal RNA gene.

Identification of Thieno[3,2‐b]Pyrrole Derivatives as Novel Small Molecule Inhibitors of Neurotropic Alphaviruses

Neurotropic alphaviruses such as western, eastern, and Venezuelan equine encephalitis viruses cause serious and potentially fatal central nervous system infections in humans and are high-priority potential bioterrorism agents. There are currently no widely available vaccines or licensed therapies for these virulent pathogens. To identify potential novel antiviral drugs, we developed a cell-based assay with a western equine encephalitis virus replicon that expresses a luciferase reporter gene and screened a small molecule diversity library of 51,028 compounds. We identified and validated a thieno[3,2-b]pyrrole compound with a half maximal inhibitory concentration of <10 micromol/L, a selectivity index>20, and potent activity against live virus in cultured neuronal cells. Furthermore, a structure-activity relationship analysis with 20 related compounds identified several with enhanced activity profiles, including 6 with submicromolar half maximal inhibitory concentrations. In conclusion, we have identified a novel class of promising inhibitors with potent activity against virulent neurotropic alphaviruses.

Urinary tract infection due to Enterobacter sakazakii

Enterobacter sakazakii is a rare but important cause of necrotizing enterocolitis, bloodstream infection and central nervous system infections in humans, with mortality rates of 40-80%. It has not been reported to cause urinary tract infection. We report a case of urinary tract infection due to E. sakazakii in a 63-year-old lady with chronic renal failure.

Systemic aspergillosis caused by an aflatoxin-producing strain of Aspergillus flavus

The first case of human systemic infection by an Aspergillus flavus isolate demonstrated to produce aflatoxins in vitro and in vivo is described. The patient, a 41-year-old man with acute myelogenous leukaemia, developed a complication of suspected pulmonary Aspergillus infection during remission induction therapy. Antifungal chemotherapy brought about a considerable degree of improvement, but remission of the underlying disease was not attained. Bone marrow transplantation was also not effective. The patient showed recovery from neutropenia but died despite aggresive antifungal chemotherapy. The autopsy revealed lesions in the lungs, myocardium, kidneys, brain, thyroid gland and skin due to a suspected Aspergillus sp. A fungus isolated from the right lung and the skin lesions was identified as A. flavus. Aflatoxins B1, B2 and M1 were detected in culture filtrates of the isolated A. flavus, and in an extract of lung lesions. These aflatoxins are considered to have played an important role in damaging the immune system of the patient through their toxic effects.

In vitro culture, serologic and molecular analysis of Acanthamoeba isolated from the liver of a keel-billed toucan ( Ramphastos sulfuratus)

Members of the genus Acanthamoeba are usually free-living amebae and are found in a variety of ecological niches including soil, fresh and brackish water, dust in air, filters of heating, ventilating, and air conditioning units, swimming pools and hot tubs, etc. Occasionally, they are also known to cause central nervous system infections in humans and other animals. We isolated into culture an amoeba from the liver tissue of a keel-billed toucan and identified it as Acanthamoeba sp. based on culture characteristics and immunofluorescent analysis. Further, we characterized the cultured amoeba and also the amoeba in the liver tissue as Acanthamoeba, genotype T4, by sequencing a diagnostic region of the nuclear small subunit ribosomal RNA gene.

Food and probiotic strains from the Saccharomyces cerevisiae species as a possible origin of human systemic infections

We report four cases of blood cultures testing positive for yeast strains belonging to the species Saccharomyces cerevisiae. Using molecular techniques, RFLP of mtDNA and δ-PCR amplification, we show the association of two of the isolates with non-clinical strains. Specifically, with two commercial bread-making strains and the therapeutic S. boulardii strain. The association of S. boulardii with cases of fungemia has been reported previously. Nevertheless, this is the first time that a baker's yeast has been isolated from blood.

Genomic sequencing of deer tick virus and phylogeny of powassan-related viruses of North America.

Powassan (POW) virus is responsible for central nervous system infection in humans in North America and the eastern parts of Russia. Recently, a new flavivirus, deer tick (DT) virus, related to POW virus was isolated in the United States, but neither its pathogenic potential in human nor the taxonomic relationship with POW virus has been elucidated. In this study, we obtained the near-full-length genomic sequence of the DT virus and complete sequences of 3 genomic regions of 15 strains of POW-related virus strains. The phylogeny revealed 2 lineages, one of which had the prototype POW virus and the other DT virus. Both lineages can cause central nervous system infection in humans. By use of the combination of molecular definition of virus species within the genus Flavivirus and serological distinction in a 2-way cross-neutralization test, the lineage of DT virus is classified as a distinct genotype of POW virus.