Shaman Muradrasoli

Environmental levels of the antiviral oseltamivir induce development of resistance mutation H274Y in influenza A/H1N1 virus in mallards.

Oseltamivir (Tamiflu®) is the most widely used drug against influenza infections and is extensively stockpiled worldwide as part of pandemic preparedness plans. However, resistance is a growing problem and in 2008–2009, seasonal human influenza A/H1N1 virus strains in most parts of the world carried the mutation H274Y in the neuraminidase gene which causes resistance to the drug. The active metabolite of oseltamivir, oseltamivir carboxylate (OC), is poorly degraded in sewage treatment plants and surface water and has been detected in aquatic environments where the natural influenza reservoir, dabbling ducks, can be exposed to the substance. To assess if resistance can develop under these circumstances, we infected mallards with influenza A/H1N1 virus and exposed the birds to 80 ng/L, 1 µg/L and 80 µg/L of OC through their sole water source. By sequencing the neuraminidase gene from fecal samples, we found that H274Y occurred at 1 µg/L of OC and rapidly dominated the viral population at 80 µg/L. IC50 for OC was increased from 2–4 nM in wild-type viruses to 400–700 nM in H274Y mutants as measured by a neuraminidase inhibition assay. This is consistent with the decrease in sensitivity to OC that has been noted among human clinical isolates carrying H274Y. Environmental OC levels have been measured to 58–293 ng/L during seasonal outbreaks and are expected to reach µg/L-levels during pandemics. Thus, resistance could be induced in influenza viruses circulating among wild ducks. As influenza viruses can cross species barriers, oseltamivir resistance could spread to human-adapted strains with pandemic potential disabling oseltamivir, a cornerstone in pandemic preparedness planning. We propose surveillance in wild birds as a measure to understand the resistance situation in nature and to monitor it over time. Strategies to lower environmental levels of OC include improved sewage treatment and, more importantly, a prudent use of antivirals.

Prevalence and phylogeny of coronaviruses in wild birds from the Bering Strait area (Beringia)

Coronaviruses (CoVs) can cause mild to severe disease in humans and animals, their host range and environmental spread seem to have been largely underestimated, and they are currently being investigated for their potential medical relevance. Infectious bronchitis virus (IBV) belongs to gamma-coronaviruses and causes a costly respiratory viral disease in chickens. The role of wild birds in the epidemiology of IBV is poorly understood. In the present study, we examined 1,002 cloacal and faecal samples collected from 26 wild bird species in the Beringia area for the presence of CoVs, and then we performed statistical and phylogenetic analyses. We detected diverse CoVs by RT-PCR in wild birds in the Beringia area. Sequence analysis showed that the detected viruses are gamma-coronaviruses related to IBV. These findings suggest that wild birds are able to carry gamma-coronaviruses asymptomatically. We concluded that CoVs are widespread among wild birds in Beringia, and their geographic spread and frequency is higher than previously realised. Thus, Avian CoV can be efficiently disseminated over large distances and could be a genetic reservoir for future emerging pathogenic CoVs. Considering the great animal health and economic impact of IBV as well as the recent emergence of novel coronaviruses such as SARS-coronavirus, it is important to investigate the role of wildlife reservoirs in CoV infection biology and epidemiology.

Broadly targeted multiprobe QPCR for detection of coronaviruses: Coronavirus is common among mallard ducks (Anas platyrhynchos)

Abstract Coronaviruses (CoVs) can cause trivial or fatal disease in humans and in animals. Detection methods for a wide range of CoVs are needed, to understand viral evolution, host range, transmission and maintenance in reservoirs. A new concept, “Multiprobe QPCR”, which uses a balanced mixture of competing discrete non- or moderately degenerated nuclease degradable (TaqMan®) probes was employed. It provides a broadly targeted and rational single tube real-time reverse transcription PCR (“NQPCR”) for the generic detection and discovery of CoV. Degenerate primers, previously published, and the new probes, were from a conserved stretch of open reading frame 1b, encoding the replicase. This multiprobe design reduced the degree of probe degeneration, which otherwise decreases the sensitivity, and allowed a preliminary classification of the amplified sequence directly from the QPCR trace. The split probe strategy allowed detection of down to 10 viral nucleic acid equivalents of CoV from all known CoV groups. Evaluation was with reference CoV strains, synthetic targets, human respiratory samples and avian fecal samples. Infectious-Bronchitis-Virus (IBV)-related variants were found in 7 of 35 sample pools, from 100 wild mallards (Anas platyrhynchos). Ducks may spread and harbour CoVs. NQPCR can detect a wide range of CoVs, as illustrated for humans and ducks.

Resistance Mutation R292K Is Induced in Influenza A(H6N2) Virus by Exposure of Infected Mallards to Low Levels of Oseltamivir

Resistance to neuraminidase inhibitors (NAIs) is problematic as these drugs constitute the major treatment option for severe influenza. Extensive use of the NAI oseltamivir (Tamiflu®) results in up to 865 ng/L of its active metabolite oseltamivir carboxylate (OC) in river water. There one of the natural reservoirs of influenza A, dabbling ducks, can be exposed. We previously demonstrated that an influenza A(H1N1) virus in mallards (Anas platyrhynchos) exposed to 1 µg/L of OC developed oseltamivir resistance through the mutation H274Y (N2-numbering). In this study, we assessed the resistance development in an A(H6N2) virus, which belongs to the phylogenetic N2 group of neuraminidases with distinct functional and resistance characteristics. Mallards were infected with A(H6N2) while exposed to 120 ng/L, 1.2 µg/L or 12 µg/L of OC in their sole water source. After 4 days with 12 µg/L of OC exposure, the resistance mutation R292K emerged and then persisted. Drug sensitivity was decreased ≈13,000-fold for OC and ≈7.8-fold for zanamivir. Viral shedding was similar when comparing R292K and wild-type virus indicating sustained replication and transmission. Reduced neuraminidase activity and decrease in recovered virus after propagation in embryonated hen eggs was observed in R292K viruses. The initial, but not the later R292K isolates reverted to wild-type during egg-propagation, suggesting a stabilization of the mutation, possibly through additional mutations in the neuraminidase (D113N or D141N) or hemagglutinin (E216K). Our results indicate a risk for OC resistance development also in a N2 group influenza virus and that exposure to one NAI can result in a decreased sensitivity to other NAIs as well. If established in influenza viruses circulating among wild birds, the resistance could spread to humans via re-assortment or direct transmission. This could potentially cause an oseltamivir-resistant pandemic; a serious health concern as preparedness plans rely heavily on oseltamivir before vaccines can be mass-produced.

Isolation and Molecular Characterization of Brucella Isolates in Cattle Milk in Uganda

Brucellosis is endemic in livestock and humans in Uganda and its transmission involves a multitude of risk factors like consumption of milk from infected cattle. To shed new light on the epidemiology of brucellosis in Uganda the present study used phenotypic and molecular approaches to delineate the Brucella species, biovars, and genotypes shed in cattle milk. Brucella abortus without a biovar designation was isolated from eleven out of 207 milk samples from cattle in Uganda. These isolates had a genomic monomorphism at 16 variable number tandem repeat (VNTR) loci and showed in turn high levels of genetic variation when compared with other African strains or other B. abortus biovars from other parts of the world. This study further highlights the usefulness of MLVA as an epidemiological tool for investigation of Brucella infections.

Oseltamivir-Resistant Influenza A (H1N1) Virus Strain with an H274Y Mutation in Neuraminidase Persists without Drug Pressure in Infected Mallards

ABSTRACT Influenza A virus (IAV) has its natural reservoir in wild waterfowl, and emerging human IAVs often contain gene segments from avian viruses. The active drug metabolite of oseltamivir (oseltamivir carboxylate [OC]), stockpiled as Tamiflu for influenza pandemic preparedness, is not removed by conventional sewage treatment and has been detected in river water. There, it may exert evolutionary pressure on avian IAV in waterfowl, resulting in the development of resistant viral variants. A resistant avian IAV can circulate among wild birds only if resistance does not restrict viral fitness and if the resistant virus can persist without continuous drug pressure. In this in vivo mallard (Anas platyrhynchos) study, we tested whether an OC-resistant avian IAV (H1N1) strain with an H274Y mutation in the neuraminidase (NA-H274Y) could retain resistance while drug pressure was gradually removed. Successively infected mallards were exposed to decreasing levels of OC, and fecal samples were analyzed for the neuraminidase sequence and phenotypic resistance. No reversion to wild-type virus was observed during the experiment, which included 17 days of viral transmission among 10 ducks exposed to OC concentrations below resistance induction levels. We conclude that resistance in avian IAV that is induced by exposure of the natural host to OC can persist in the absence of the drug. Thus, there is a risk that human-pathogenic IAVs that evolve from IAVs circulating among wild birds may contain resistance mutations. An oseltamivir-resistant pandemic IAV would pose a substantial public health threat. Therefore, our observations underscore the need for prudent oseltamivir use, upgraded sewage treatment, and surveillance for resistant IAVs in wild birds.

Broadly targeted triplex real-time PCR detection of influenza A, B and C viruses based on the nucleoprotein gene and a novel “MegaBeacon” probe strategy

Abstract A PCR assay that covers animal and human influenza A, B and C viruses, i.e., most of Orthomyxoviridae, is needed. Influenza types are distinguished based on differences in the nucleoprotein (NP) present in the virus. Conserved NP regions were therefore used to design a TaqMan®-based triplex reverse transcription real-time PCR method. Variability of influenza A within the probe target region mandated the development of a novel molecular beacon, the “Mega” molecular beacon (MegaBeacon; MegB), for the detection of influenza A with this method. MegaBeacon is a mismatch-tolerant molecular beacon that is also a TaqMan® probe. The triplex method (3QPCR-MegB) was evaluated with influenza A isolates covering 18 HxNx combinations, two influenza B isolates, and five Japanese influenza C isolates, as well as influenza A, B and C synthetic DNA targets. One to ten viral RNA and cDNA genome equivalents were detected per PCR reaction for influenza A, B and C. Seventy-one human nasopharyngeal aspirates from respiratory infections yielded 30 influenza A, 11 influenza B and 0 influenza C with 3QPCR-MegB, where immunofluorescence (IF) found 28 influenza A and 10 influenza B. 3QPCR-MegB was more mismatch-tolerant than a variant PCR with an influenza A TaqMan® probe (3QPCR) and is a sensitive and rational method to detect influenza viruses of animal and human origin. MegaBeacon probes hold promise for variable target nucleic acids.

Influenza A(H7N9) Virus Acquires Resistance-Related Neuraminidase I222T Substitution When Infected Mallards Are Exposed to Low Levels of Oseltamivir in Water

ABSTRACT Influenza A virus (IAV) has its natural reservoir in wild waterfowl, and new human IAVs often contain gene segments originating from avian IAVs. Treatment options for severe human influenza are principally restricted to neuraminidase inhibitors (NAIs), among which oseltamivir is stockpiled in preparedness for influenza pandemics. There is evolutionary pressure in the environment for resistance development to oseltamivir in avian IAVs, as the active metabolite oseltamivir carboxylate (OC) passes largely undegraded through sewage treatment to river water where waterfowl reside. In an in vivo mallard (Anas platyrhynchos) model, we tested if low-pathogenic avian influenza A(H7N9) virus might become resistant if the host was exposed to low levels of OC. Ducks were experimentally infected, and OC was added to their water, after which infection and transmission were maintained by successive introductions of uninfected birds. Daily fecal samples were tested for IAV excretion, genotype, and phenotype. Following mallard exposure to 2.5 μg/liter OC, the resistance-related neuraminidase (NA) I222T substitution, was detected within 2 days during the first passage and was found in all viruses sequenced from subsequently introduced ducks. The substitution generated 8-fold and 2.4-fold increases in the 50% inhibitory concentration (IC50) for OC (P < 0.001) and zanamivir (P = 0.016), respectively. We conclude that OC exposure of IAV hosts, in the same concentration magnitude as found in the environment, may result in amino acid substitutions, leading to changed antiviral sensitivity in an IAV subtype that can be highly pathogenic to humans. Prudent use of oseltamivir and resistance surveillance of IAVs in wild birds are warranted.

Murine Gammaretrovirus Group G3 Was Not Found in Swedish Patients with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome and Fibromyalgia

Background The recent report of gammaretroviruses of probable murine origin in humans, called xenotropic murine retrovirus related virus (XMRV) and human murine leukemia virus related virus (HMRV), necessitated a bioinformatic search for this virus in genomes of the mouse and other vertebrates, and by PCR in humans. Results Three major groups of murine endogenous gammaretroviruses were identified. The third group encompassed both exogenous and endogenous Murine Leukemia Viruses (MLVs), and most XMRV/HMRV sequences reported from patients suffering from myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Two sensitive real-time PCRs for this group were developed. The predicted and observed amplification range for these and three published XMRV/HMRV PCRs demonstrated conspicuous differences between some of them, partly explainable by a recombinatorial origin of XMRV. Three reverse transcription real-time PCRs (RTQPCRs), directed against conserved and not overlapping stretches of env, gag and integrase (INT) sequences of XMRV/HMRV were used on human samples. White blood cells from 78 patients suffering from ME/CFS, of which 30 patients also fulfilled the diagnostic criteria for fibromyalgia (ME/CFS/FM) and in 7 patients with fibromyalgia (FM) only, all from the Gothenburg area of Sweden. As controls we analyzed 168 sera from Uppsala blood donors. We controlled for presence and amplifiability of nucleic acid and for mouse DNA contamination. To score as positive, a sample had to react with several of the XMRV/HMRV PCRs. None of the samples gave PCR reactions which fulfilled the positivity criteria. Conclusions XMRV/HMRV like proviruses occur in the third murine gammaretrovirus group, characterized here. PCRs developed by us, and others, approximately cover this group, except for the INT RTQPCR, which is rather strictly XMRV specific. Using such PCRs, XMRV/HMRV could not be detected in PBMC and plasma samples from Swedish patients suffering from ME/CFS/FM, and in sera from Swedish blood donors.

Comparison of two H1N2 swine influenza A viruses from disease outbreaks in pigs in Sweden during 2009 and 2010.

The influenza A virus subtypes H1N1, H1N2 and H3N2 are prevalent in pig populations worldwide. In the present study, two relatively uncommon swine influenza virus (SIV) H1N2 subtypes, isolated in Sweden in 2009 and 2010, were compared regarding their molecular composition and biological characteristics. The differences regarding markers purportedly related to pathogenicity, host adaptation or replication efficiency. They included a truncated PB1-F2 protein in the earlier isolate but a full length version in the more recent one; differences in the number of haemagglutinin glycosylation sites, including a characteristic human one; and a nuclear export protein with altered export signal. Of particular interest, the NS1 amino acid sequence of swine H1N2-2009 and 2010 has a ‘unique or very unusual’ PDZ binding domain (RPKV) at the C-terminal of the protein, a motif that has been implicated as a virulence marker. Concerning biological properties, these viruses reached lower titre and showed reduced cytopathogenicity in MDCK cells compared with an avian-like H1N1 isolate A/swine/Lidkoping/1193/2002 belonging to the same lineage as the 2009 and 2010 isolates. The findings should contribute to better understanding of factors related to the survival/extinction of this uncommon reassortant variant.