Description: Currently, nitritation-anammox (anaerobic ammonium oxidation) bioreactors are designed to treat wastewaters with high ammonium concentrations at mesophilic temperatures (25 to 40°C). The implementation of this technology at ambient temperatures for nitrogen removal from municipal wastewater following carbon removal may lead to more-sustainable technology with energy and cost savings. However, the application of nitritation-anammox bioreactors at low temperatures (characteristic of municipal wastewaters except in tropical and subtropical regions) has not yet been explored. To this end, a laboratory-scale (5-liter) nitritation-anammox sequencing batch reactor was adapted to 12°C in 10 days and operated for more than 300 days to investigate the feasibility of nitrogen removal from synthetic pretreated municipal wastewater by the combination of aerobic ammonium-oxidizing bacteria (AOB) and anammox. The activities of both anammox and AOB were high enough to remove more than 90% of the supplied nitrogen. Multiple aspects, including the presence and activity of anammox, AOB, and aerobic nitrite oxidizers (NOB) and nitrous oxide (N 2 O) emission, were monitored to evaluate the stability of the bioreactor at 12°C. There was no nitrite accumulation throughout the operational period, indicating that anammox bacteria were active at 12°C and that AOB and anammox bacteria outcompeted NOB. Moreover, our results showed that sludge from wastewater treatment plants designed for treating high-ammonium-load wastewaters can be used as seeding sludge for wastewater treatment plants aimed at treating municipal wastewater that has a low temperature and low ammonium concentrations.

Description: Nonnucleoside reverse transcriptase (RT) inhibitors (NNRTI) and integrase (IN) strand transfer inhibitors (INSTI) are key components of antiretroviral regimens. To explore potential interactions between NNRTI and INSTI resistance mutations, we investigated the combined effects of these mutations on drug susceptibility and fitness of human immunodeficiency virus type 1 (HIV-1). In the absence of drug, single-mutant viruses were less fit than the wild type; viruses carrying multiple mutations were less fit than single-mutant viruses. These findings were explained in part by the observation that mutant viruses carrying NNRTI plus INSTI resistance mutations had reduced amounts of virion-associated RT and/or IN protein. In the presence of efavirenz (EFV), a virus carrying RT-K103N together with IN-G140S and IN-Q148H (here termed IN-G140S/Q148H) mutations was fitter than a virus with a RT-K103N mutation alone. Similarly, in the presence of EFV, the RT-E138K plus IN-G140S/Q148H mutant virus was fitter than one with the RT-E138K mutation alone. No effect of INSTI resistance mutations on the fitness of RT-Y181C mutant viruses was observed. Conversely, RT-E138K and -Y181C mutations improved the fitness of the IN-G140S/Q148H mutant virus in the presence of raltegravir (RAL); the RT-K103N mutation had no effect. The NNRTI resistance mutations had no effect on RAL susceptibility. Likewise, the IN-G140S/Q148H mutations had no effect on EFV or RPV susceptibility. However, both the RT-K103N plus IN-G140S/Q148H and the RT-E138K plus IN-G140S/Q148H mutant viruses had significantly greater fold increases in 50% inhibitory concentration (IC 50 ) of EFV than viruses carrying a single NNRTI mutation. Likewise, the RT-E138K plus IN-G140S/Q148H mutant virus had significantly greater fold increases in RAL IC 50 than that of the IN-G140S/Q148H mutant virus. These results suggest that interactions between RT and IN mutations are important for NNRTI and INSTI resistance and viral fitness. IMPORTANCE Nonnucleoside reverse transcriptase inhibitors and integrase inhibitors are used to treat infection with HIV-1. Mutations that confer resistance to these drugs reduce the ability of HIV-1 to reproduce (that is, they decrease viral fitness). It is known that reverse transcriptase and integrase interact and that some mutations can disrupt their interaction, which is necessary for proper functioning of these two enzymes. To determine whether resistance mutations in these enzymes interact, we investigated their effects on drug sensitivity and viral fitness. Although individual drug resistance mutations usually reduced viral fitness, certain combinations of mutations increased fitness. When present in certain combinations, some integrase inhibitor resistance mutations increased resistance to nonnucleoside reverse transcriptase inhibitors and vice versa. Because these drugs are sometimes used together in the treatment of HIV-1 infection, these interactions could make viruses more resistant to both drugs, further limiting their clinical benefit.

Description: We sought to identify and characterize microRNA (miRNAs) that posttranscriptionally regulate the expression of scavenger receptor class B type I (SR-BI) and SR-BI-linked selective high-density lipoprotein (HDL) cholesteryl ester (CE) transport and steroidogenesis. Four miRNAs (miRNA-125a, miRNA-125b, miRNA-145, and miRNA-455) with a potential to regulate SR-BI were identified in silico and validated by quantitative real-time PCR (qRT-PCR), Western blot analysis, and SR-BI 3' untranslated region (UTR) reporter assays. In vitro treatment of primary rat granulosa cells and MLTC-1 cells with cyclic AMP (cAMP) or in vivo treatment of rat adrenals with adrenocorticotropic hormone (ACTH) decreased the expression of miRNA-125a, miRNA-125b, and miRNA-455 and reciprocally increased SR-BI expression. Using luciferase constructs containing the 3' untranslated region of SR-BI combined with miRNA overexpression and mutagenesis, we have provided evidence that steroidogenic SR-BI is a direct target of miRNA-125a and miRNA-455. Moreover, the transfection of Leydig tumor cells with precursor miRNA 125a (pre-miRNA-125a) or pre-miRNA-455 resulted in the suppression of SR-BI at both the transcript and protein levels and reduced selective HDL CE uptake and HDL-stimulated progesterone production. Transfection of liver Hepa 1-6 cells with pre-miRNA-125a significantly reduced SR-BI expression and its selective transport function. In contrast, overexpression of miRNA-145 did not affect SR-BI expression or selective HDL CE uptake mediated by SR-BI in steroidogenic cell lines. These data suggest that a trophic hormone and cAMP inversely regulate the expression of SR-BI and miRNA-125a and miRNA-455 in steroidogenic tissues/cells and that both miRNA-125a and miRNA-455, by targeting steroidogenic SR-BI, negatively regulate selective HDL CE uptake and HDL CE-supported steroid hormone production.

Description: Highly pathogenic H5N1 influenza A virus remains a substantial threat to public health. To understand the molecular basis and host mechanism for the high virulence of H5N1 viruses in mammals, we compared two H5N1 isolates which have similar genetic backgrounds but greatly differ in their virulence in mice. A/Chicken/Jiangsu/k0402/2010 (CK10) is highly pathogenic, whereas A/Goose/Jiangsu/k0403/2010 (GS10) is nonpathogenic. We first showed that CK10 elicited a more potent innate immune response than did GS10 in mouse lungs by increasing the number and expression levels of activated genes. We then generated a series of reassortants between the two viruses and evaluated their virulence in mice. Inclusion of the CK10 PA gene in the GS10 background resulted in a dramatic increase in virulence. Conversely, expression of the GS10 PA gene in the CK10 background significantly attenuated the virulence. These results demonstrated that the PA gene mainly determines the pathogenicity discrepancy between CK10 and GS10 in mice. We further determined that arginine (R) at position 353 of the PA gene contributes to the high virulence of CK10 in mice. The reciprocal substitution at position 353 in PA or the exchange of the entire PA gene largely caused the transfer of viral phenotypes, including virus replication, polymerase activity, and manipulation of the innate response, between CK10 and GS10. We therefore defined a novel molecular marker associated with the high virulence of H5N1 influenza viruses, providing further insights into the pathogenesis of H5N1 viruses in mammals.

Description: A total of 146 group B streptococcus isolates from 8 cities across China belonged to four serotypes. Serotype Ia was more common in children. A high prevalence of resistance was observed for levofloxacin (37.7%), erythromycin (71.2%), clindamycin, (53.4%), and tetracycline (81.5%). The levofloxacin and clindamycin resistances among the 4 serotypes differed significantly. Eighty percent of fluoroquinolone-resistant isolates belonged to the sequence type 19 (ST19)/serotype III clone, with GyrA-ParC-ParE triple substitutions. This clone carried the erm (B), mef (E), and tet (M) genes.

Description: Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf75 ( ac75 ) is a highly conserved gene of unknown function. In this study, we constructed an ac75 knockout AcMNPV bacmid and investigated the role of ac75 in the baculovirus life cycle. The expression and distribution of the Ac75 protein were characterized, and its interaction with another viral protein was analyzed to further understand its function. Our data indicated that ac75 was required for the nuclear egress of nucleocapsids, intranuclear microvesicle formation, and subsequent budded virion (BV) formation, as well as occlusion-derived virion (ODV) envelopment and embedding of ODVs into polyhedra. Western blot analyses showed that two forms, of 18 and 15 kDa, of FLAG-tagged Ac75 protein were detected. Ac75 was associated with both nucleocapsid and envelope fractions of BVs but with only the nucleocapsid fraction of ODVs; the 18-kDa form was associated with only BVs, whereas the 15-kDa form was associated with both types of virion. Ac75 was localized predominantly in the intranuclear ring zone during infection and exhibited a nuclear rim distribution during the early phase of infection. A phase separation assay suggested that Ac75 was not an integral membrane protein. A coimmunoprecipitation assay revealed an interaction between Ac75 and the integral membrane protein Ac76, and bimolecular fluorescence complementation assays identified the sites of the interaction within the cytoplasm and at the nuclear membrane and ring zone in AcMNPV-infected cells. Our results have identified ac75 as a second gene that is required for both the nuclear egress of nucleocapsids and the formation of intranuclear microvesicles. IMPORTANCE During the baculovirus life cycle, the morphogenesis of both budded virions (BVs) and occlusion-derived virions (ODVs) is proposed to involve a budding process at the nuclear membrane, which occurs while nucleocapsids egress from the nucleus or when intranuclear microvesicles are produced. However, the exact mechanism of virion morphogenesis remains unknown. In this study, we identified ac75 as a second gene, in addition to ac93 , that is essential for the nuclear egress of nucleocapsids, intranuclear microvesicle formation, and subsequent BV formation, as well as ODV envelopment and embedding of ODVs into polyhedra. Ac75 is not an integral membrane protein. However, it interacts with an integral membrane protein (Ac76) and is associated with the nuclear membrane. These data enhance our understanding of the commonalities between nuclear egress of nucleocapsids and intranuclear microvesicle formation and may help to reveal insights into the mechanism of baculovirus virion morphogenesis.

Description: Most highly pathogenic avian influenza A viruses cause only mild clinical signs in ducks, serving as an important natural reservoir of influenza A viruses. However, we isolated two H5N1 viruses that are genetically similar but differ greatly in virulence in ducks. A/Chicken/Jiangsu/k0402/2010 (CK10) is highly pathogenic, whereas A/Goose/Jiangsu/k0403/2010 (GS10) is low pathogenic. To determine the genetic basis for the high virulence of CK10 in ducks, we generated a series of single-gene reassortants between CK10 and GS10 and tested their virulence in ducks. Expression of the CK10 PA or hemagglutinin (HA) gene in the GS10 context resulted in increased virulence and virus replication. Conversely, inclusion of the GS10 PA or HA gene in the CK10 background attenuated the virulence and virus replication. Moreover, the PA gene had a greater contribution. We further determined that residues 101G and 237E in the PA gene contribute to the high virulence of CK10. Mutations at these two positions produced changes in virulence, virus replication, and polymerase activity of CK10 or GS10. Position 237 plays a greater role in determining these phenotypes. Moreover, the K237E mutation in the GS10 PA gene increased PA nuclear accumulation. Mutant GS10 viruses carrying the CK10 HA gene or the PA101G or PA237E mutation induced an enhanced innate immune response. A sustained innate response was detected in the brain rather than in the lung and spleen. Our results suggest that the PA and HA gene-mediated high virus replication and the intense innate immune response in the brain contribute to the high virulence of H5N1 virus in ducks.

Description: The nucleoprotein (N) and phosphoprotein (P) interaction of nonsegmented negative-strand RNA viruses is essential for viral replication; this includes N 0 -P (N 0 , free of RNA) interaction and the interaction of N-RNA with P. The precise site(s) within N that mediates the N-P interaction and the detailed regulating mechanism, however, are less clear. Using a human parainfluenza virus type 3 (HPIV3) minigenome assay, we found that an N mutant (N L478A ) did not support reporter gene expression. Using in vivo and in vitro coimmunoprecipitation, we found that N L478A maintains the ability to form N L478A 0 -P, to self-assemble, and to form N L478A -RNA but that N L478A -RNA does not interact with P. Using an immunofluorescence assay, we found that N-P interaction provides the minimal requirement for the formation of cytoplasmic inclusion bodies, which contain viral RNA, N, P, and polymerase in HPIV3-infected cells. N L478A was unable to form inclusion bodies when coexpressed with P, but the presence of N rescued the ability of N L478A to form inclusion bodies and the transcriptional function of N L478A , thereby suggesting that hetero-oligomers formed by N and N L478A are functional and competent to form inclusion bodies. Furthermore, we found that N L478A is also defective in virus growth. To our knowledge, we are the first to use a paramyxovirus to identify a precise amino acid within N that is critical for N-RNA and P interaction but not for N 0 -P interaction for the formation of inclusion bodies, which appear to be bona fide sites of RNA synthesis.

Description: The entry of baculovirus budded virus into host cells is mediated by two distinct types of envelope fusion proteins (EFPs), GP64 and F protein. Phylogenetic analysis suggested that F proteins were ancestral baculovirus EFPs, whereas GP64 was acquired by progenitor group I alphabaculovirus more recently and may have stimulated the formation of the group I lineage. This study was designed to experimentally recapitulate a possible major step in the evolution of baculoviruses. We demonstrated that the infectivity of an F-null group II alphabaculovirus ( Helicoverpa armigera nucleopolyhedrovirus [HearNPV]) can be functionally rescued by coinsertion of GP64 along with the nonfusogenic F def (furin site mutated HaF) from HearNPV. Interestingly, HearNPV enters cells by endocytosis and, less efficiently, by direct membrane fusion at low pH. However, this recombinant HearNPV coexpressing F def and GP64 mimicked group I virus not only in its EFP composition but also in its abilities to enter host cells via low-pH-triggered direct fusion pathway. Neutralization assays indicated that the nonfusogenic F proteins contribute mainly to binding to susceptible cells, while GP64 contributes to fusion. Coinsertion of GP64 with an F-like protein (Ac23) from group I virus led to efficient rescue of an F-null group II virus. In summary, these recombinant viruses and their entry modes are considered to resemble an evolutionary event of the acquisition of GP64 by an ancestral group I virus and subsequent adaptive inactivation of the original F protein. The study described here provides the first experimental evidence to support the hypothesis of the evolution of baculovirus EFPs.

Description: Therapy for hepatitis C virus (HCV) infection has advanced with the recent approval of direct-acting antivirals in combination with peginterferon and ribavirin. New antivirals with novel targets are still needed to further improve the treatment of hepatitis C. Previously reported screening methods for HCV inhibitors either are limited to a virus-specific function or apply a screening method at a single dose, which usually leads to high false-positive or -negative rates. We developed a quantitative high-throughput screening (qHTS) assay platform with a cell-based HCV infection system. This highly sensitive assay can be miniaturized to a 1,536-well format for screening of large chemical libraries. All candidates are screened over a 7-concentration dose range to give EC 50 s (compound concentrations at 50% efficacy) and dose-response curves. Using this assay format, we screened a library of pharmacologically active compounds (LOPAC). Based on the profile of dose-dependent curves of HCV inhibition and cytotoxicity, 22 compounds with adequate curves and EC 50 s of 〈10 μM were selected for validation. In two additional independent assays, 17 of them demonstrated specific inhibition of HCV infection. Ten potential candidates with efficacies of 〉70% and CC 50 s (compound concentrations at 50% cytotoxicity) of 〈30 μM from these validated hits were characterized for their target stages in the HCV replication cycle. In this screen, we identified both known and novel hits with diverse structural and functional features targeting various stages of the HCV replication cycle. The pilot screen demonstrates that this assay system is highly robust and effective in identifying novel HCV inhibitors and that it can be readily applied to large-scale screening of small-molecule libraries.