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Shipping influenza virus specimens, isolates or purified RNA is normally conducted at ultra-low temperatures using dry ice to ensure minimal degradation of the samples but this is expensive and requires special packaging and shipping conditions. Therefore, alternative methods for shipping influenza viruses or RNA at ambient temperatures would be desirable. The RNASound RNA Sampling Card (FortiusBio LLC, San Diego, CA, USA) is a device that enables specimens or isolates to be applied to a card, whereby viruses are inactivated, while RNA is preserved and purified RNA can also easily be eluted. To evaluate this card, we applied influenza virus cell culture isolate supernatants to either the RNASound card or Whatman Grade No. 1 filter paper (GE Healthcare, Rydalmere, NSW, Australia) and compared the preservation to that of material stored in liquid form. Preservation was tested using influenza A and B viruses at two different storage temperatures [cool (2–8∘C) or room temperature (18–22∘C)] and these were compared with control material stored at -80∘C, for 7, 14, or 28 days. The quality of the RNA recovered was assessed using real time RT-PCR and Sanger sequencing. The RNASound card was effective in preserving influenza RNA at room temperature for up to 28 days, with only a minor change in real-time RT-PCR cycle threshold values for selected gene targets when comparing between viruses applied to the card or stored at -80∘C. Similar results were obtained with filter paper, whilst virus in liquid form performed the worst. Nevertheless, as the RNASound card also has the capability to inactivate viruses in addition to preserving RNA at room temperature for many weeks, this makes it feasible to send samples to laboratories using regular mail, and thus avoid the need for expensive shipping conditions requiring biohazard containers and dry ice. Moreover, the quick and simple RNA recovery from the RNASound card allows recipient labs to obtain RNA without the need for special reagents or equipment. [ABSTRACT FROM AUTHOR]

Understanding the spatiotemporal patterns of emergence and circulation of new human seasonal influenza virus variants is a key scientific and public health challenge. The global circulation patterns of influenza A/H3N2 viruses are well characterized, but the patterns of A/H1N1 and B viruses have remained largely unexplored. Here we show that the global circulation patterns of A/H1N1 (up to 2009), B/Victoria, and B/Yamagata viruses differ substantially from those of A/H3N2 viruses, on the basis of analyses of 9,604 haemagglutinin sequences of human seasonal influenza viruses from 2000 to 2012. Whereas genetic variants of A/H3N2 viruses did not persist locally between epidemics and were reseeded from East and Southeast Asia, genetic variants of A/H1N1 and B viruses persisted across several seasons and exhibited complex global dynamics with East and Southeast Asia playing a limited role in disseminating new variants. The less frequent global movement of influenza A/H1N1 and B viruses coincided with slower rates of antigenic evolution, lower ages of infection, and smaller, less frequent epidemics compared to A/H3N2 viruses. Detailed epidemic models support differences in age of infection, combined with the less frequent travel of children, as probable drivers of the differences in the patterns of global circulation, suggesting a complex interaction between virus evolution, epidemiology, and human behaviour. [ABSTRACT FROM AUTHOR]

Objectives: The burden of disease due to influenza B is often underestimated. Clinical studies have shown that oseltamivir, a widely used neuraminidase inhibitor (NAI) antiviral drug, may have reduced effectiveness against influenza B viruses. Therefore, it is important to study the effect of neuraminidasemutations in influenza B viruses that may further reduce NAI susceptibility, and to determine whether these mutations have the same effect in the two lineages of influenza B viruses that are currently circulating (B/Yamagata-like and B/Victoria-like). Methods:We characterized the effect of 16 amino acid substitutions across five framework residues and four monomeric interface residues on the susceptibility to four different NAIs (oseltamivir, zanamivir, peramivir and laninamivir). Results: Framework residue mutations E117A and E117G conferred highly reduced inhibition to three of the four NAIs, but substantially reduced neuraminidase activity, whereas other framework mutations retained a greater level of NA activity. Mutations E105K, P139S and G140R of themonomeric interfacewere also found to cause highly reduced inhibition, but, interestingly, their effect was substantially greater in a B/Victoria-like neuraminidase than in a B/Yamagata-like neuraminidase, with some susceptibility values being up to 1000-fold different between lineages. Conclusions: The frequency and the effect of key neuraminidase mutations on neuraminidase activity and NAI susceptibility can differ substantially between the two influenza B lineages. Therefore, future surveillance, analysis and interpretation of influenza B virus NAI susceptibility should consider the B lineage of the neuraminidase in the same manner as already occurs for different influenza A neuraminidase subtypes. [ABSTRACT FROM AUTHOR]

This review highlights the main points which emerged from the presentations and discussions at the 3rd isirv- Antiviral Group Conference - advances in clinical management. The conference covered emerging and potentially pandemic influenza viruses and discussed novel/pre-licensure therapeutics and currently approved antivirals and vaccines for the control of influenza. Current data on approved and novel treatments for non-influenza respiratory viruses such as MERS- Co V, respiratory syncytial virus ( RSV) and rhinoviruses and the challenges of treating immunocompromised patients with respiratory infections was highlighted. [ABSTRACT FROM AUTHOR]

Objectives: The emergence of the pandemic influenza A(H1N1)pdm09 virus in 2009 saw a significant increase in the therapeutic and prophylactic use of neuraminidase inhibitors (NAIs) to mitigate the impact of this highly transmissible virus. Prior to the pandemic, many countries stockpiled NAIs and developed pandemic plans for the use of antiviral drugs, based on either treatment of high-risk individuals and/or prophylaxis of contacts. However, to date there has been a lack of in vivo models to test the efficacy of treatment or prophylaxis with NAIs, for influenza-infected individuals or exposed contacts, in a household setting.Methods: A ferret model of household contact was developed to study the efficacy of different prophylaxis regimens in preventing infection in contact ferrets exposed to influenza A(H1N1)pdm09-infected index ferrets.Results: Among the different prophylactic regimens, contact ferrets receiving oseltamivir prophylaxis twice daily showed better outcomes than those receiving oseltamivir once daily. Benefits included a significant delay in the time to secondary infection, lower weight loss and higher activity levels. The treatment of index ferrets at 36 h post-infection did not influence either secondary infection rates or clinical symptoms in exposed contact ferrets. Neither prophylaxis nor treatment prevented infection or reduced the duration of viral shedding, although clinical symptoms did improve in infected animals receiving prophylaxis.Conclusions: Different oseltamivir prophylaxis regimens did not prevent infections, but consistently resulted in a reduction in symptoms in infected ferrets. However, oseltamivir prophylaxis failed to reduce viral titres, which warrants further investigation in humans. [ABSTRACT FROM AUTHOR]

Influenza viruses collected from regions of Asia, Africa and Oceania between 2009 and 2012 were tested for their susceptibility to two new neuraminidase inhibitors, peramivir and laninamivir. All viruses tested had normal laninamivir inhibition. However, 3·2% (19/599) of A(H1N1)pdm09 viruses had highly reduced peramivir inhibition (due to H275Y NA mutation) and <1% (6/1238) of influenza B viruses had reduced or highly reduced peramivir inhibition, with single occurrence of variants containing I221T, A245T, K360E, A395E, D432G and a combined G145R+Y142H mutation. These data demonstrate that despite an increase in H275Y variants in 2011, there was no marked change in the frequency of peramivir- or laninamivir-resistant variants following the market release of the drugs in Japan in 2010. [ABSTRACT FROM AUTHOR]

Antivirals play an important role in the prevention and treatment of influenza infections, particularly in high-risk or severely ill patients. Two classes of influenza antivirals have been available inmany countries over the last decade (2004-2013), the adamantanes and the neuraminidase inhibitors (NAIs). During this period, widespread adamantane resistance has developed in circulating influenza viruses rendering these drugs useless, resulting in the reliance on the most widely available NAI, oseltamivir. However, the emergence of oseltamivir-resistant seasonal A(H1N1) viruses in 2008 demonstrated that NAI-resistant viruses could also emerge and spread globally in a similarmanner to that seen for adamantane-resistant viruses. Previously, it was believed that NAI-resistant viruses had compromised replication and/or transmission. Fortunately, in 2013, themajority of circulating human influenza viruses remain sensitive to all of the NAIs, but significant work by our laboratory and others is now underway to understand what enables NAI-resistant viruses to retain the capacity to replicate and transmit. In this review, we describe how the susceptibility of circulating human and avian influenza viruses has changed over the last ten years and describe some research studies that aim to understand how NAI-resistant human and avian influenza viruses may emerge in the future. [ABSTRACT FROM AUTHOR]

A minor viral population of oseltamivir-resistant A( H3 N2) viruses ( E119 V neuraminidase mutation) was selected and maintained in a continually infected immunocompromised child following initial oseltamivir treatment. A subsequent course of oseltamivir given 7 weeks later rapidly selected for the E119 V variant resulting in a near-pure population of the resistant virus. The study highlights the challenges of oseltamivir treatment of immunocompromised patients that are continually shedding virus and demonstrates the ability of the E119 V oseltamivir-resistant virus to be maintained for prolonged periods even in the absence of drug-selective pressure. [ABSTRACT FROM AUTHOR]

Please cite this paper as: Kelly et al. (2012) The significance of increased influenza notifications during spring and summer of 2010-11 in Australia. Influenza and Other Respiratory Viruses. DOI: 10.1111/irv.12057. Background & objective During the temperate out-of-season months in Australia in late 2010 and early 2011, an unprecedented high number of influenza notifications were recorded. We aimed to assess the significance of these notifications. Methods For Australia, we used laboratory-confirmed cases notified to the WHO FluNet surveillance tool; the percentage of these that were positive; notifications by state and influenza type and subtype; and surveillance data from Google FluTrends. For the state of Victoria, we used laboratory-confirmed notified cases and influenza-like illness (ILI) proportions. We compared virus characterisation using haemagglutination-inhibition assays and phylogenetic analysis of the haemagglutinin gene for seasonal and out-of-season notifications. Results The increase in notifications was most marked in tropical and subtropical Australia, but the number of out-of-season notifications in temperate Victoria was more than five times higher than the average of the previous three seasons. However, ILI proportions in spring-summer were not different to previous years. All out-of-season viruses tested were antigenically and genetically similar to those tested during either the 2010 or 2011 influenza seasons. An increase in the number of laboratories testing for influenza has led to an increase in the number of tests performed and cases notified. Conclusion An increase in influenza infections in spring-summer of 2010-11 in tropical and temperate Australia was not associated with any differences in virus characterisation compared with viruses that circulated in the preceding and following winters. This increase probably reflected a natural variation in out-of-season virus circulation, which was amplified by increased laboratory testing. [ABSTRACT FROM AUTHOR]

Influenza A and B viruses cause significant morbidity and mortality worldwide each year. The neuraminidase inhibitors (NAIs) are the most commonly used class of influenza antiviral drugs for the treatment of infected patients. In vitro studies have shown that influenza B viruses are significantly less susceptible to oseltamivir and other neuraminidase inhibitors compared with influenza A viruses. Following analysis of published clinical studies, we show that oseltamivir does appear to have lower effectiveness in patients infected with influenza B virus compared with influenza A infected patients, but due to insufficient studies on zanamivir, laninamivir or peramivir, it was not possible to conclude the relative effectiveness of these drugs against influenza A virus compared with B virus. [ABSTRACT FROM AUTHOR]

Summary: Influenza A H1N1 2009 virus caused the first pandemic in an era when neuraminidase inhibitor antiviral drugs were available in many countries. The experiences of detecting and responding to resistance during the pandemic provided important lessons for public health, laboratory testing, and clinical management. We propose recommendations for antiviral susceptibility testing, reporting results, and management of patients infected with 2009 pandemic influenza A H1N1. Sustained global monitoring for antiviral resistance among circulating influenza viruses is crucial to inform public health and clinical recommendations for antiviral use, especially since community spread of oseltamivir-resistant A H1N1 2009 virus remains a concern. Further studies are needed to better understand influenza management in specific patient groups, such as severely immunocompromised hosts, including optimisation of antiviral treatment, rapid sample testing, and timely reporting of susceptibility results. [Copyright &y& Elsevier]

Analysis of mutations I117V and I117M in the neuraminidase of influenza A pandemic (H1N1) 2009 viruses showed that I117V confers a mild reduction in oseltamivir sensitivity and has a synergistic effect of further increasing resistance when combined with H275Y. Contrary to recent reports, the I117M mutation does not alter oseltamivir sensitivity. [ABSTRACT FROM AUTHOR]

The 2010 Victorian influenza season was characterized by normal seasonal influenza activity and the dominance of the pandemic A(H1N1) 2009 strain. General Practice Sentinel Surveillance rates peaked at 9.4 ILI cases per 1000 consultations in week 36 for metropolitan practices, and at 10.5 ILI cases per 1000 in the following week for rural practices. Of the 678 ILI cases, 23% were vaccinated, a significantly higher percentage than in previous years. A significantly higher percentage of ILI patients were swabbed in 2010 compared to 2003-2008, but similar to 2009, with a similar percentage being positive for influenza as in previous years. Vaccination rates increased with patient age. Melbourne Medical Deputising Service rates peaked in week 35 at 19.1 ILI cases per 1000 consultations. Of the 1914 cases of influenza notified to the Department of Health, Victoria, 1812 (95%) were influenza A infections - 1001 (55%) pandemic A(H1N1) 2009, 4 (<1%) A(H3N2) and 807 (45%) not subtyped; 88 (5%) were influenza B; and 14 (<1%) were influenza A and B co-infections. The World Health Organization Collaborating Centre for Reference and Research on Influenza tested 403 isolates of which 261 were positive for influenza, 250 of which were influenza A and 11 were influenza B. Ninety-two per cent of the influenza A viruses were pandemic A(H1N1) 2009, and following antigenic analysis all of these were found to be similar to the current vaccine strain. Three viruses (0.9%) were found to be oseltamivir resistant due to an H275Y mutation in the neuraminidase gene. [ABSTRACT FROM AUTHOR]

We investigated carriage of avian influenza viruses by wild birds in Australia, 2005-2008, to assess the risks to poultry industries and human health. We collected 21,858 (7,357 cloacal, 14,501 fecal) samples and detected 300 viruses, representing a detection rate of =1.4%. Rates were highest in autumn (March-May) and differed substantially between bird types, areas, and years. We typed 107 avian influenza viruses and identified 19 H5, 8 H7, and 16 H9 (40% of typed viruses). All were of low pathogenicity. These viruses formed clearly different phylogenetic clades to lineages from Eurasia or North America, suggesting the potential existence of Australian lineages. H7 viruses were similar to highly pathogenic H7 strains that caused outbreaks in poultry in Australia. Several periods of increased detection rates (numbers or subtypes of viruses) were identified. This study demonstrates the need for ongoing surveillance to detect emerging pathogenic strains and facilitate prevention of outbreaks. [ABSTRACT FROM AUTHOR]

Abstract: Using an in vivo ferret model, we investigated the development of resistance to oseltamivir and zanamivir for two different influenza A(H5N1) viruses (A/Vietnam/1203/2004, haemagglutinin phylogenetic clade 1, and A/Chicken/Laos/26/2006, haemagglutinin phylogenetic clade 2.3) by treating the animals with doses equivalent either to the recommended human treatment dose or a range of sub-optimal drug doses. No resistance was observed in oseltamivir-treated ferrets, but analysis of nasal washes from zanamivir-treated ferrets infected with influenza A/Vietnam/1203/2004 revealed one viral isolate (from a ferret receiving the highest dose of zanamivir, 1.0mg/kg twice daily) with a zanamivir IC50 that was 350-fold higher than the other isolates tested. The same virus also demonstrated a 26-fold increase in oseltamivir IC50. The isolate with reduced susceptibility was taken from a ferret 8 days post-infection that was being treated with the recommended human zanamivir dose. Sequence analysis of the resistant virus revealed a glutamine (Q) to leucine (L) mutation at residue 136 of the neuraminidase. This is the first report of this mutation being associated with neuraminidase inhibitor susceptibility and one of the few reported mutations that confer zanamivir resistance, and as such should be closely monitored in influenza A(H5N1) and other N1 viruses in the future. Further animal studies and human clinical trials are necessary to optimize neuraminidase inhibitor dosing strategies for the treatment of influenza A(H5N1) infections. [ABSTRACT FROM AUTHOR]

Influenza outbreaks during mass gatherings have been rarely described, and detailed virologic assessment is lacking. An influenza outbreak occurred during World Youth Day in Sydney, Australia, July 2008 (WYD2008), We assessed epidemiologic data and respiratory samples collected from attendees who sought treatment for influenza-like illness at emergency clinics in Sydney during this outbreak. Isolated influenza viruses were compared with seasonal influenza viruses from the 2008 influenza season. From 100 infected attendees, numerous strains were identified: oseltamivir-resistant influenza A (H1N1) viruses, oseltamivir-sensitive influenza A (H1N1) viruses, influenza A (H3N2) viruses, and strains from both influenza B lineages (B/FIorida/4/2006-1ike and B/Malaysia/2506/2004-like). Novel viruses were introduced, and pre-WYD2008 seasonal viruses were amplified. Viruses isolated at mass gatherings can have substantial, complex, and unpredictable effects on community influenza activity, Greater flexibility by public health authorities and hospitals is required to appropriately manage and contain these outbreaks. [ABSTRACT FROM AUTHOR]

During the 2007 equine influenza outbreak in Australia, respiratory disease in dogs in close contact with infected horses was noted; influenza (H3N8) virus infection was confirmed. Nucleotide sequence of the virus from dogs was identical to that from horses. No evidence of dog-to-dog transmission or virus persistence in dogs was found. [ABSTRACT FROM AUTHOR]

Along with influenza vaccines, the world is currently almost completely dependent on two licensed drugs for the treatment or prevention of seasonal (influenza A and B viruses) and pandemic influenza (influenza A viruses). These drugs - oseltamivir (Tamiflu®) and zanamivir (Relenza®) - are classified as neuraminidase inhibitors (NAIs) because they act by inhibiting one of the key surface proteins of the influenza virus, the neuraminidase, which in turn reduces the ability of the virus to infect other respiratory cells. Our dependence on these drugs has arisen because of high levels of resistance with seasonal influenza viruses to the older class of anti-influenza drugs, the adamantanes (amantadine and rimantadine), combined with the lack of activity of these drugs against influenza B viruses. Recently, however, significant levels of oseltamivir-resistant influenza A(H1) seasonal influenza viruses have also been encountered, which has been associated with a single amino acid change in the viral neuraminidase (H274Y). Oseltamivir is the most widely used and stockpiled NAI and, while these A(H1) viruses are still sensitive to zanamivir, it highlights the ease with which the influenza virus can mutate and reassort to circumvent available drugs. Fortunately, the current pandemic A(H1N1) 2009 virus, which is circulating globally, remains largely sensitive to both NAIs, although a small number of oseltamivir-resistant viruses have been isolated from patients to date, again with the H274Y mutation. Clearly there is a need to use the NAI drugs prudently to ensure they remain an effective defence against future seasonal and pandemic influenza viruses, along with careful monitoring of levels of resistance in the circulating viruses combined with the further development of new antiinfluenza drugs. [ABSTRACT FROM AUTHOR]