Characterization of Influenza B Virus Variants with Reduced Neuraminidase Inhibitor Susceptibility.

Treatment options for influenza B virus infections are limited to neuraminidase inhibitors (NAIs), which block the neuraminidase (NA) glycoprotein on the virion surface. The development of NAI resistance would therefore result in a loss of antiviral treatment options for influenza B virus infections. This study characterized two contemporary influenza B viruses with known resistance-conferring NA amino acid substitutions, D197N and H273Y, detected during routine surveillance. The D197N and H273Y variants were characterized in vitro by assessing NA enzyme activity and affinity, as well as replication in cell culture compared to those of NAI-sensitive wild-type viruses. In vivo studies were also performed in ferrets to assess the replication and transmissibility of each variant. Mathematical models were used to analyze within-host and between-host fitness of variants relative to wild-type viruses. The data revealed that the H273Y variant had NA enzyme function similar to that of its wild type but had slightly reduced replication and transmission efficiency in vivo The D197N variant had impaired NA enzyme function, but there was no evidence of reduction in replication or transmission efficiency in ferrets. Our data suggest that the influenza B virus variant with the H273Y NA substitution had a more notable reduction in fitness compared to wild-type viruses than the influenza B variant with the D197N NA substitution. Although a D197N variant is yet to become widespread, it is the most commonly detected NAI-resistant influenza B virus in surveillance studies. Our results highlight the need to carefully monitor circulating viruses for the spread of influenza B viruses with the D197N NA substitution.

Characteristics of a widespread community cluster of H275Y oseltamivir-resistant A(H1N1)pdm09 influenza in Australia.

BACKGROUND: Oseltamivir resistance in A(H1N1)pdm09 influenza is rare, particularly in untreated community cases. Sustained community transmission has not previously been reported. METHODS: Influenza specimens from the Asia-Pacific region were collected through sentinel surveillance, hospital, and general practitioner networks. Clinical and epidemiological information was collected on patients infected with oseltamivir-resistant viruses. RESULTS: Twenty-nine (15%) of 191 A(H1N1)pdm09 viruses collected between May and September 2011 from Hunter New England (HNE), Australia, contained the H275Y neuraminidase substitution responsible for oseltamivir resistance. Only 1 patient had received oseltamivir before specimen collection. The resistant strains were genetically very closely related, suggesting the spread of a single variant. Ninety percent of cases lived within 50 kilometers. Three genetically similar oseltamivir-resistant variants were detected outside of HNE, including 1 strain from Perth, approximately 4000 kilometers away. Computational analysis predicted that neuraminidase substitutions V241I, N369K, and N386S in these viruses may offset the destabilizing effect of the H275Y substitution. CONCLUSIONS: This cluster represents the first widespread community transmission of H275Y oseltamivir-resistant A(H1N1)pdm09 influenza. These cases and data on potential permissive mutations suggest that currently circulating A(H1N1)pdm09 viruses retain viral fitness in the presence of the H275Y mutation and that widespread emergence of oseltamivir-resistant strains may now be more likely.

Oseltamivir-resistant influenza A(H1N1)pdm09 virus in Dutch travellers returning from Spain, August 2012.

Two Dutch travellers were infected with oseltamivir-resistant influenza A(H1N1)pdm09 viruses with an H275Y neuraminidase substitution in early August 2012. Both cases were probably infected during separate holidays at the Catalonian coast (Spain). No epidemiological connection between the two cases was found, and neither of them was treated with oseltamivir before specimen collection. Genetic analysis of the neuraminidase gene revealed the presence of previously described permissive mutations that may increase the likelihood of such strains emerging and spreading widely.

Increased detection in Australia and Singapore of a novel influenza A(H1N1)2009 variant with reduced oseltamivir and zanamivir sensitivity due to a S247N neuraminidase mutation.

A novel influenza A(H1N1)2009 variant with mildly reduced oseltamivir and zanamivir sensitivity has been detected in more than 10% of community specimens in Singapore and more than 30% of samples from northern Australia during the early months of 2011. The variant, which has also been detected in other regions of the Asia-Pacific, contains a S247N neuraminidase mutation. When combined with the H275Y mutation, as detected in an oseltamivir-treated patient, the dual S247N+H275Y mutant had extremely high oseltamivir resistance.

Oseltamivir-resistant influenza viruses circulating during the first year of the influenza A(H1N1) 2009 pandemic in the Asia-Pacific region, March 2009 to March 2010.

During the first year of the influenza A(H1N1) 2009 pandemic, unprecedented amounts of the neuraminidase inhibitors, predominantly oseltamivir, were used in economically developed countries for the treatment and prophylaxis of patients prior to the availability of a pandemic vaccine. Due to concerns about the development of resistance, over 1,400 influenza A(H1N1) 2009 viruses isolated from the Asia-Pacific region during the first year of the pandemic (March 2009 to March 2010) were analysed by phenotypic and genotypic assays to determine their susceptibility to the neuraminidase inhibitors. Amongst viruses submitted to the World Health Organization Collaborating Centre for Reference and Research in Melbourne, Australia,oseltamivir resistance was detected in 1.3% of influenza A(H1N1) 2009 strains from Australia and 3.1% of strains from Singapore, but none was detected in specimens received from other countries in Oceania or south-east Asia, or in east Asia. The overall frequency of oseltamivir resistance in the Asia-Pacific region was 16 of 1,488 (1.1%). No zanamivir-resistant viruses were detected. Of the 16 oseltamivir-resistant isolates detected, nine were from immunocompromised individuals undergoing oseltamivir treatment and three were from immunocompetent individuals undergoing oseltamivir treatment. Importantly, four oseltamivir-resistant strains were from immunocompetent individuals who had not been treated with oseltamivir, demonstrating limited low-level community transmission of oseltamivir-resistant strains. Even with increased use of oseltamivir during the pandemic, the frequency of resistance has been low, with little evidence of community-wide spread of the resistant strains. Nevertheless, prudent use of the neuraminidase inhibitors remains necessary, as does continued monitoring for drug-resistant influenza viruses.

Characterization of substitutions in the neuraminidase of A(H7N9) influenza viruses selected following serial passage in the presence of different neuraminidase inhibitors.

Avian A(H7N9) infections in humans have been reported in China since 2013 and are of public health concern due to their severity and pandemic potential. Oseltamivir and peramivir are neuraminidase inhibitors (NAIs) routinely used for the treatment of A(H7N9) infections, but variants with reduced sensitivity to these drugs can emerge in patients during treatment. Zanamivir and laninamivir are NAIs that are used less frequently. Herein, we performed in vitro serial passaging experiments with recombinant viruses, containing the neuraminidase (NA) from influenza A/Anhui/1/13 (H7N9) virus, in the presence of each NAI, to determine whether variants with reduced sensitivity would emerge. NA substitutions were characterized for their effect on the NA enzymatic activity and surface expression of the A/Anhui/1/13 (Anhui/1) NA, as well as NAs originating from contemporary A(H7N9) viruses of the Yangtze River Delta and Pearl River Delta lineages. In vitro passage in the presence of oseltamivir, peramivir and laninamivir selected for substitutions associated with reduced sensitivity (E119D, R292K and R152K), whereas passage in the presence of zanamivir did not select for any viruses with reduced sensitivity. All the NA substitutions significantly reduced activity, but not the expression of the Anhui/1 NA. In contemporary N9 NAs, all substitutions tested significantly reduced NA enzyme function in the Yangtze River lineage background, but not in the Pearl River Delta lineage background. Overall, these findings suggest that zanamivir may be less likely than the other NAIs to select for resistance in A(H7N9) viruses and that the impact of substitutions that reduce NAI susceptibility or enzyme function may be less in A(H7N9) viruses from the Pearl River lineage.

The emergence of adamantane resistance in influenza A(H1) viruses in Australia and regionally in 2006.

The adamantanes (amantadine and rimantadine) were the first antivirals licensed for use against influenza A viruses and have been used in some countries to control seasonal influenza. While increasing resistance of A(H3) viruses to this class of drug has been reported in recent years, only low levels of resistance were seen with A(H1) viruses until the 2005-2006 influenza season in the USA. In this study we analysed 101 human influenza A viruses isolated in 2006 that were referred to the WHO Collaborating Centre for Reference and Research in Melbourne, from Australia and the surrounding regions, for evidence of resistance to adamantanes. We found that whereas previously A(H1) resistant viruses were rare, 21.8% of the 2006 viruses had a resistant genotype. By comparison, 58.6% of influenza A(H3) viruses isolated in 2006 that were tested at the Centre, had a resistant genotype.

Increased adamantane resistance in influenza A(H3) viruses in Australia and neighbouring countries in 2005.

The prevention and control of disease caused by seasonal and potential pandemic influenza viruses is currently managed by the use influenza vaccines and antivirals. The adamantanes (amantadine and rimantadine) were the first antivirals licensed for use against influenza A viruses and have been used extensively in some countries. Since the early 2000s increased resistance to these drugs has been reported especially in the A(H3) viruses. In this study we analysed recent human influenza A strains isolated in Australia and regionally for evidence of resistance to adamantanes and found evidence of significant resistant emerging during 2005.

Susceptibility of highly pathogenic A(H5N1) avian influenza viruses to the neuraminidase inhibitors and adamantanes.

Since 2003, highly pathogenic A(H5N1) influenza viruses have been the cause of large-scale death in poultry and the subsequent infection and death of over 140 humans. A group of 55 influenza A(H5N1) viruses isolated from various regions of South East Asia between 2004 and 2006 were tested for their susceptibility to the anti-influenza drugs the neuraminidase inhibitors and adamantanes. The majority of strains were found to be fully sensitive to the neuraminidase inhibitors oseltamivir carboxylate, zanamivir and peramivir; however two strains demonstrated increased IC50 values. Sequence analysis of these strains revealed mutations in the normally highly conserved residues 116 and 117 of the N1 neuraminidase. Sequence analysis of the M2 gene showed that all of the A(H5N1) viruses from Vietnam, Malaysia and Cambodia contained mutations (L26I and S31N) associated with resistance to the adamantane drugs (rimantadine and amantadine), while strains from Indonesia were found to be a mix of both adamantane resistant (S31N) and sensitive viruses. None of the A(H5N1) viruses from Myanmar contained mutations known to confer adamantane resistance. These results support the use of neuraminidase inhibitors as the most appropriate class of antiviral drug to prevent or treat human A(H5N1) virus infections.

Avian influenza in Australia: a summary of 5 years of wild bird surveillance.

BACKGROUND: Avian influenza viruses (AIVs) are found worldwide in numerous bird species, causing significant disease in gallinaceous poultry and occasionally other species. Surveillance of wild bird reservoirs provides an opportunity to add to the understanding of the epidemiology of AIVs. METHODS: This study examined key findings from the National Avian Influenza Wild Bird Surveillance Program over a 5-year period (July 2007-June 2012), the main source of information on AIVs circulating in Australia. RESULTS: The overall proportion of birds that tested positive for influenza A via PCR was 1.9 ± 0.1%, with evidence of widespread exposure of Australian wild birds to most low pathogenic avian influenza (LPAI) subtypes (H1-13, H16). LPAI H5 subtypes were found to be dominant and widespread during this 5-year period. CONCLUSION: Given Australia's isolation, both geographically and ecologically, it is important for Australia not to assume that the epidemiology of AIV from other geographic regions applies here. Despite all previous highly pathogenic avian influenza outbreaks in Australian poultry being attributed to H7 subtypes, widespread detection of H5 subtypes in wild birds may represent an ongoing risk to the Australian poultry industry.

Antibody landscapes after influenza virus infection or vaccination.

We introduce the antibody landscape, a method for the quantitative analysis of antibody-mediated immunity to antigenically variable pathogens, achieved by accounting for antigenic variation among pathogen strains. We generated antibody landscapes to study immune profiles covering 43 years of influenza A/H3N2 virus evolution for 69 individuals monitored for infection over 6 years and for 225 individuals pre- and postvaccination. Upon infection and vaccination, titers increased broadly, including previously encountered viruses far beyond the extent of cross-reactivity observed after a primary infection. We explored implications for vaccination and found that the use of an antigenically advanced virus had the dual benefit of inducing antibodies against both advanced and previous antigenic clusters. These results indicate that preemptive vaccine updates may improve influenza vaccine efficacy in previously exposed individuals.

Infection of dogs with equine influenza virus: evidence for transmission from horses during the Australian outbreak.

During the equine influenza (EI) outbreak, respiratory disease was observed in dogs that were in close proximity to infected horses. Investigations were undertaken to exclude influenza virus infection. Of the 23 dogs that were seropositive in tests using the influenza A/Sydney/2007 virus as the test antigen, 10 showed clinical signs. EI virus appeared to be readily transmitted to dogs that were held in close proximity to infected horses, but there was no evidence of lateral transmission of the virus to other dogs that did not have contact with or were not held in close proximity to horses.

Adamantane resistance in influenza A(H1) viruses increased in 2007 in South East Asia but decreased in Australia and some other countries.

The adamantanes (amantadine and rimantadine) were the initial antivirals licensed for use against influenza A viruses and have been used in some countries to control seasonal influenza and have also been stockpiled for potential pandemic use. While high rates of resistance have been observed in recent years with A(H3) viruses, the rates of resistance with A(H1) viruses has varied widely. In this study we analysed 281 human influenza A viruses isolated in 2007 that were referred to the WHO Collaborating Centre for Reference and Research in Melbourne, mainly from Australia and the surrounding regions, for evidence of resistance to adamantanes and a subset of these was examined for resistance to the neuraminidase inhibitors (NIs). We found that the rates of adamantane resistance in A(H3) viruses continued to increase in most countries in 2007 but a distinct variation was seen with A(H1) resistance levels. A(H1) viruses from Australia, New Zealand and Europe had low rates of resistance (2-9%) whereas viruses from a number of South East (SE) Asian countries had high rates of resistance (33-100%). This difference can be attributed to the spread of A/Brisbane/59/2007-like viruses to many parts of the world with the exception of SE Asia where A/Hong Kong/2652/2006-like viruses continue to predominate. When these two A(H1) subgroups were compared for their in vitro sensitivity to the other class of influenza antiviral drugs, the neuraminidase inhibitors, no difference was seen between the groups with both showing normal levels of sensitivity to these drugs, The finding of reducing A(H1) resistance rates in Australia and rising levels in SE Asia in 2007, reverses the trend seen in 2006 when A(H1) resistance levels were rising in Australia and elsewhere but remained low in most of SE Asia.

Genetic analysis of two influenza A (H1) swine viruses isolated from humans in Thailand and the Philippines.

Influenza viruses A/Philippines/341/2004 (H1N2) and A/Thailand/271/2005 (H1N1) were isolated from two males, with mild influenza providing evidence of sporadic human infection by contemporary swine influenza. Both viruses were antigenically and genetically distinct from influenza A (H1N1 and H1N2) viruses that have circulated in the human population. Genetic analysis of the haemagglutinin genes found these viruses to have the highest degree of similarity to the classical swine H1 viruses circulating in Asia and North America. The neuraminidase gene and the internal genes were found to be more closely related to viruses circulating in European swine, which appear to have undergone multiple reassorting events. Although transmission of swine influenza to humans appears to be a relatively rare event, swine have been proposed as the intermediate host in the generation of potential pandemic influenza virus that may have the capacity to cause human epidemics resulting in high morbidity and mortality.

Isolation of avian influenza viruses from two different transhemispheric migratory shorebird species in Australia.

Shorebirds on their southerly migration from Siberia to Australia, may pass through Asian regions currently experiencing outbreaks of highly pathogenic H5N1 influenza. To test for the presence of avian influenza viruses in migratory shorebirds arriving in Australia during spring 2004, 173 cloacal swabs were collected from six species. Ten swabs were positive for influenza A, with H4N8 viruses detected in five red-necked stints and H11N9 viruses detected in five sharp-tailed sandpipers. No H5N1 viruses were detected. All isolated viruses were non-pathogenic in domestic chickens. These results further demonstrate the potential for migratory shorebirds to carry and potentially spread influenza viruses.

An influenza A(H3) reassortant was epidemic in Australia and New Zealand in 2003.

During 2003, Australia and New Zealand experienced substantial outbreaks of influenza. The strain responsible was an A(H3N2) influenza virus described as A/Fujian/411/2002-like, which had circulated as a minor variant in the previous Northern Hemisphere (NH) winter, mainly in Korea and Japan. Early in the year the isolates were very similar to those that had been previously isolated in the NH, however, a reassortant strain emerged early in the New Zealand winter, followed by the appearance of similar viruses in Australia and other regional areas. While the hemagglutinin HA1 sequence of these viruses demonstrated only minor differences from the A/Fujian/411/2002 reference strain, the neuraminidase gene was clearly different from that of other recently circulating H3 viruses and most closely matched an earlier reference strain A/Chile/6416/2001. Three internal genes (NS, NP, M) in the reassortant viruses were also more closely related to the A/Chile/6416/2001 lineage. This reassortant A(H3) virus predominated in Australia and New Zealand in 2003 was also seen in Brazil and Malaysia during 2003 and was widespread in the United States and Europe during their 2003-04 winter. Interestingly most of the strains of A(H3) that were isolated at the beginning of the 2004 winter in Australia, did not have this earlier A/Chile/6416/2001-like neuraminidase but had a neuraminidase that was similar to that of the reference strain A/Fujian/411/2002. This was suggestive of the re-introduction of influenza A(H3) from other countries, however, there was still low level circulation of the reassortant virus in 2004 with isolates detected in Australia and Singapore.