Host Cell Copper Transporters CTR1 and ATP7A are important for Influenza A virus replication.

BACKGROUND: The essential role of copper in eukaryotic cellular physiology is known, but has not been recognized as important in the context of influenza A virus infection. In this study, we investigated the effect of cellular copper on influenza A virus replication. METHODS: Influenza A/WSN/33 (H1N1) virus growth and macromolecule syntheses were assessed in cultured human lung cells (A549) where the copper concentration of the growth medium was modified, or expression of host genes involved in copper homeostasis was targeted by RNA interference. RESULTS: Exogenously increasing copper concentration, or chelating copper, resulted in moderate defects in viral growth. Nucleoprotein (NP) localization, neuraminidase activity assays and transmission electron microscopy did not reveal significant defects in virion assembly, morphology or release under these conditions. However, RNAi knockdown of the high-affinity copper importer CTR1 resulted in significant viral growth defects (7.3-fold reduced titer at 24 hours post-infection, p = 0.04). Knockdown of CTR1 or the trans-Golgi copper transporter ATP7A significantly reduced polymerase activity in a minigenome assay. Both copper transporters were required for authentic viral RNA synthesis and NP and matrix (M1) protein accumulation in the infected cell. CONCLUSIONS: These results demonstrate that intracellular copper regulates the influenza virus life cycle, with potentially distinct mechanisms in specific cellular compartments. These observations provide a new avenue for drug development and studies of influenza virus pathogenesis.

Disparities in capreomycin resistance levels associated with the rrs A1401G mutation in clinical isolates of Mycobacterium tuberculosis.

As the prevalence of multidrug-resistant and extensively drug-resistant tuberculosis strains continues to rise, so does the need to develop accurate and rapid molecular tests to complement time-consuming growth-based drug susceptibility testing. Performance of molecular methods relies on the association of specific mutations with phenotypic drug resistance and while considerable progress has been made for resistance detection of first-line antituberculosis drugs, rapid detection of resistance for second-line drugs lags behind. The rrs A1401G allele is considered a strong predictor of cross-resistance between the three second-line injectable drugs, capreomycin (CAP), kanamycin, and amikacin. However, discordance is often observed between the rrs A1401G mutation and CAP resistance, with up to 40% of rrs A1401G mutants being classified as CAP susceptible. We measured the MICs to CAP in 53 clinical isolates harboring the rrs A1401G mutation and found that the CAP MICs ranged from 8 µg/ml to 40 µg/ml. These results were drastically different from engineered A1401G mutants generated in isogenic Mycobacterium tuberculosis, which exclusively exhibited high-level CAP MICs of 40 µg/ml. These data support the results of prior studies, which suggest that the critical concentration of CAP (10 µg/ml) used to determine resistance by indirect agar proportion may be too high to detect all CAP-resistant strains and suggest that a larger percentage of resistant isolates could be identified by lowering the critical concentration. These data also suggest that differences in resistance levels among clinical isolates are possibly due to second site or compensatory mutations located elsewhere in the genome.

Filament-producing mutants of influenza A/Puerto Rico/8/1934 (H1N1) virus have higher neuraminidase activities than the spherical wild-type.

Influenza virus exhibits two morphologies - spherical and filamentous. Strains that have been grown extensively in laboratory substrates are comprised predominantly of spherical virions while clinical or low passage isolates produce a mixture of spheres and filamentous virions of varying lengths. The filamentous morphology can be lost upon continued passage in embryonated chicken eggs, a common laboratory substrate for influenza viruses. The fact that the filamentous morphology is maintained in nature but lost in favor of a spherical morphology in ovo suggests that filaments confer a selective advantage within the infected host that is not necessary for growth in laboratory substrates. Indeed, we have recently shown that filament-producing variant viruses are selected upon passage of the spherical laboratory strain A/Puerto Rico/8/1934 (H1N1) [PR8] in guinea pigs. Toward determining the nature of the selective advantage conferred by filaments, we sought to identify functional differences between spherical and filamentous particles. We compared the wild-type PR8 virus to two previously characterized recombinant PR8 viruses in which single point mutations within M1 confer a filamentous morphology. Our results indicate that these filamentous PR8 mutants have higher neuraminidase activities than the spherical PR8 virus. Conversely, no differences were observed in HAU:PFU or HAU:RNA ratios, binding avidity, sensitivity to immune serum in hemagglutination inhibition assays, or virion stability at elevated temperatures. Based on these results, we propose that the pleomorphic nature of influenza virus particles is important for the optimization of neuraminidase functions in vivo.

Residue 41 of the Eurasian avian-like swine influenza a virus matrix protein modulates virion filament length and efficiency of contact transmission.

UNLABELLED: Position 41 of the influenza A virus matrix protein encodes a highly conserved alanine in human and avian lineages. Nonetheless, strains of the Eurasian avian-like swine (Easw) lineage contain a change at this position: position 41 of A/swine/Spain/53207/04 (H1N1) (SPN04) encodes a proline. To assess the impact of this naturally occurring polymorphism on viral fitness, we utilized reverse genetics to produce recombinant viruses encoding wild-type M1 41P (rSPN04-P) and consensus 41A (rSPN04-A) residues. Relative to rSPN04-A, rSPN04-P virus displayed reduced growth in vitro. In the guinea pig model, rSPN04-P was transmitted to fewer contact animals than rSPN04-A and failed to infect guinea pigs that received a low-dose inoculum. Moreover, the P41A change altered virion morphology, reducing the number and length of filamentous virions, as well as reducing the neuraminidase activity of virions. The lab-adapted human isolate, A/PR/8/34 (H1N1) (PR8), is nontransmissible in the guinea pig model, making it a useful background in which to identify certain viral factors that enhance transmissibility. We assessed transmission in the context of single-, double-, and triple-reassortant viruses between PR8 and SPN04; PR8/SPN04 M, PR8/SPN04 M+NA, and PR8/SPN04 M+NA+HA, encoding either matrix 41 A or P, were generated. In each case, the virus possessing 41P transmitted less well than the corresponding 41A-encoding virus. In summary, we have identified a naturally occurring mutation in the influenza A virus matrix protein that impacts transmission efficiency and can alter virion morphology and neuraminidase activity. IMPORTANCE: We have developed a practical model for examining the genetics underlying transmissibility of the Eurasian avian-like swine lineage viruses, which contributed M and NA segments to the 2009 pandemic strain. Here, we use our system to investigate the impact on viral fitness of a naturally occurring polymorphism at matrix (M1) position 41 in an Easw isolate. Position 41 has been implicated previously in adaptation to laboratory substrates and to mice. Here we show that the polymorphism at M1 41 has a limited effect on growth in vitro but changes the morphology of the virus and impacts growth and transmission in the guinea pig model.

The M segment of the 2009 pandemic influenza virus confers increased neuraminidase activity, filamentous morphology, and efficient contact transmissibility to A/Puerto Rico/8/1934-based reassortant viruses.

UNLABELLED: The 2009 H1N1 lineage represented the first detection of a novel, highly transmissible influenza A virus genotype: six gene segments originated from the North American triple-reassortant swine lineage, and two segments, NA and M, derived from the Eurasian avian-like swine lineage. As neither parental lineage transmits efficiently between humans, the adaptations and mechanisms underlying the pandemic spread of the swine-origin 2009 strain are not clear. To help identify determinants of transmission, we used reverse genetics to introduce gene segments of an early pandemic isolate, A/Netherlands/602/2009 [H1N1] (NL602), into the background of A/Puerto Rico/8/1934 [H1N1] (PR8) and evaluated the resultant viruses in a guinea pig transmission model. Whereas the NL602 virus spread efficiently, the PR8 virus did not transmit. Swapping of the HA, NA, and M segments of NL602 into the PR8 background yielded a virus with indistinguishable contact transmissibility to the wild-type pandemic strain. Consistent with earlier reports, the pandemic M segment alone accounted for much of the improvement in transmission. To aid in understanding how the M segment might affect transmission, we evaluated neuraminidase activity and virion morphology of reassortant viruses. Transmission was found to correlate with higher neuraminidase activity and a more filamentous morphology. Importantly, we found that introduction of the pandemic M segment alone resulted in an increase in the neuraminidase activity of two pairs of otherwise isogenic PR8-based viruses. Thus, our data demonstrate the surprising result that functions encoded by the influenza A virus M segment impact neuraminidase activity and, perhaps through this mechanism, have a potent effect on transmissibility. IMPORTANCE: Our work uncovers a previously unappreciated mechanism through which the influenza A virus M segment can alter the receptor-destroying activity of an influenza virus. Concomitant with changes to neuraminidase activity, the M segment impacts the morphology of the influenza A virion and transmissibility of the virus in the guinea pig model. We suggest that changes in NA activity underlie the ability of the influenza M segment to influence virus transmissibility. Furthermore, we show that coadapted M, NA, and HA segments are required to provide optimal transmissibility to an influenza virus. The M-NA functional interaction we describe appears to underlie the prominent role of the 2009 pandemic M segment in supporting efficient transmission and may be a highly important means by which influenza A viruses restore HA/NA balance following reassortment or transfer to new host environments.

Novel H7N9 influenza virus shows low infectious dose, high growth rate, and efficient contact transmission in the guinea pig model.

The zoonotic outbreak of H7N9 subtype avian influenza virus that occurred in eastern China in the spring of 2013 resulted in 135 confirmed human cases, 44 of which were lethal. Sequencing of the viral genome revealed a number of molecular signatures associated with virulence or transmission in mammals. We report here that, in the guinea pig model, a human isolate of novel H7N9 influenza virus, A/Anhui/1/2013 (An/13), is highly dissimilar to an H7N1 avian isolate and instead behaves similarly to a human seasonal strain in several respects. An/13 was found to have a low 50% infectious dose, grow to high titers in the upper respiratory tract, and transmit efficiently among cocaged guinea pigs. The pH of fusion of the hemagglutinin (HA) and the binding of virus to fixed guinea pig tissues were also examined. The An/13 HA displayed a relatively elevated pH of fusion characteristic of many avian strains, and An/13 resembled avian viruses in terms of attachment to tissues. One important difference was seen between An/13 and both the H3N2 human and the H7N1 avian viruses: when inoculated intranasally at a high dose, only the An/13 virus led to productive infection of the lower respiratory tract of guinea pigs. In sum, An/13 was found to retain fusion and attachment properties of an avian influenza virus but displayed robust growth and contact transmission in the guinea pig model atypical of avian strains and indicative of mammalian adaptation.

Aminoglycoside cross-resistance in Mycobacterium tuberculosis due to mutations in the 5' untranslated region of whiB7.

Since the discovery of streptomycin's bactericidal activity against Mycobacterium tuberculosis, aminoglycosides have been utilized to treat tuberculosis (TB). Today, the aminoglycosides kanamycin and amikacin are used to treat multidrug-resistant (MDR) TB, and resistance to any of the second-line injectable antibiotics, including kanamycin, amikacin, or capreomycin, is a defining characteristic of extensively drug-resistant (XDR) TB. Resistance to kanamycin and streptomycin is thought to be due to the acquisition of unlinked chromosomal mutations. However, we identified eight independent mutations in the 5' untranslated region of the transcriptional activator whiB7 that confer low-level resistance to both aminoglycosides. The mutations lead to 23- to 145-fold increases in whiB7 transcripts and subsequent increased expression of both eis (Rv2416c) and tap (Rv1258c). Increased expression of eis confers kanamycin resistance in these mutants, while increased expression of tap, which encodes an efflux pump, is a previously uncharacterized mechanism of low-level streptomycin resistance. Additionally, high-level resistance to streptomycin arose at a much higher frequency in whiB7 mutants than in a wild-type (WT) strain. Although whiB7 is typically associated with intrinsic antibiotic resistance in M. tuberculosis, these data suggest that mutations in an uncharacterized regulatory region of whiB7 contribute to cross-resistance against clinically used second-line antibiotics. As drug resistance continues to develop and spread, understanding the mechanisms and molecular basis of antibiotic resistance is critical for the development of rapid molecular tests to diagnose drug-resistant TB strains and ultimately for designing regimens to treat drug-resistant cases of TB.

HIV-1 subtype C superinfected individuals mount low autologous neutralizing antibody responses prior to intrasubtype superinfection.

BACKGROUND: The potential role of antibodies in protection against intra-subtype HIV-1 superinfection remains to be understood. We compared the early neutralizing antibody (NAb) responses in three individuals, who were superinfected within one year of primary infection, to ten matched non-superinfected controls from a Zambian cohort of subtype C transmission cases. Sequence analysis of single genome amplified full-length envs from a previous study showed limited diversification in the individuals who became superinfected with the same HIV-1 subtype within year one post-seroconversion. We hypothesized that this reflected a blunted NAb response, which may have made these individuals more susceptible to superinfection. RESULTS: Neutralization assays showed that autologous plasma NAb responses to the earliest, and in some cases transmitted/founder, virus were delayed and had low to undetectable titers in all three superinfected individuals prior to superinfection. In contrast, NAbs with a median IC50 titer of 1896 were detected as early as three months post-seroconversion in non-superinfected controls. Early plasma NAbs in all subjects showed limited but variable levels of heterologous neutralization breadth. Superinfected individuals also exhibited a trend toward lower levels of gp120- and V1V2-specific IgG binding antibodies but higher gp120-specific plasma IgA binding antibodies. CONCLUSIONS: These data suggest that the lack of development of IgG antibodies, as reflected in autologous NAbs as well as gp120 and V1V2 binding antibodies to the primary infection virus, combined with potentially competing, non-protective IgA antibodies, may increase susceptibility to superinfection in the context of settings where a single HIV-1 subtype predominates.

Molecular detection of mutations associated with first- and second-line drug resistance compared with conventional drug susceptibility testing of Mycobacterium tuberculosis.

The emergence of multi- and extensively drug-resistant tuberculosis is a significant impediment to the control of this disease because treatment becomes more complex and costly. Reliable and timely drug susceptibility testing is critical to ensure that patients receive effective treatment and become noninfectious. Molecular methods can provide accurate and rapid drug susceptibility results. We used DNA sequencing to detect resistance to the first-line antituberculosis drugs isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), and ethambutol (EMB) and the second-line drugs amikacin (AMK), capreomycin (CAP), kanamycin (KAN), ciprofloxacin (CIP), and ofloxacin (OFX). Nine loci were sequenced: rpoB (for resistance to RIF), katG and inhA (INH), pncA (PZA), embB (EMB), gyrA (CIP and OFX), and rrs, eis, and tlyA (KAN, AMK, and CAP). A total of 314 clinical Mycobacterium tuberculosis complex isolates representing a variety of antibiotic resistance patterns, genotypes, and geographical origins were analyzed. The molecular data were compared to the phenotypic data and the accuracy values were calculated. Sensitivity and specificity values for the first-line drug loci were 97.1% and 93.6% for rpoB, 85.4% and 100% for katG, 16.5% and 100% for inhA, 90.6% and 100% for katG and inhA together, 84.6% and 85.8% for pncA, and 78.6% and 93.1% for embB. The values for the second-line drugs were also calculated. The size and scope of this study, in numbers of loci and isolates examined, and the phenotypic diversity of those isolates support the use of DNA sequencing to detect drug resistance in the M. tuberculosis complex. Further, the results can be used to design diagnostic tests utilizing other mutation detection technologies.

Rapid detection of multidrug-resistant Mycobacterium tuberculosis by use of real-time PCR and high-resolution melt analysis.

The current study describes the development of a unique real-time PCR assay for the detection of mutations conferring drug resistance in Mycobacterium tuberculosis. The rifampicin resistance determinant region (RRDR) of rpoB and specific regions of katG and the inhA promoter were targeted for the detection of rifampin (RIF) and isoniazid (INH) resistance, respectively. Additionally, this assay was multiplexed to discriminate Mycobacterium tuberculosis complex (MTC) strains from nontuberculous Mycobacteria (NTM) strains by targeting the IS6110 insertion element. High-resolution melting (HRM) analysis following real-time PCR was used to identify M. tuberculosis strains containing mutations at the targeted loci, and locked nucleic acid (LNA) probes were used to enhance the detection of strains containing specific single-nucleotide polymorphism (SNP) transversion mutations. This method was used to screen 252 M. tuberculosis clinical isolates, including 154 RIF-resistant strains and 174 INH-resistant strains based on the agar proportion method of drug susceptibility testing (DST). Of the 154 RIF-resistant strains, 148 were also resistant to INH and therefore classified as multidrug resistant (MDR). The assay demonstrated sensitivity and specificity of 91% and 98%, respectively, for the detection of RIF resistance and 87% and 100% for the detection of INH resistance. Overall, this assay showed a sensitivity of 85% and a specificity of 98% for the detection of MDR strains. This method provides a rapid, robust, and inexpensive way to detect the dominant mutations known to confer MDR in M. tuberculosis strains and offers several advantages over current molecular and culture-based techniques.