A 11-year-old boy with acute myeloid leukemia was brought for treatment of severe acute respiratory infection in the National Capital Region, New Delhi, India. Avian influenza A(H5N1) infection was laboratory confirmed. Complete genome analysis indicated hemagglutinin gene clade 2.3.2.1a. We found the strain to be susceptible to amantadine and neuraminidase inhibitors.

N-linked glycosylation plays critical roles in folding, receptor binding, and immunomodulating of hemagglutinin (HA), the main antigen in influenza vaccines. Chicken embryos are the predominant production host for influenza vaccines, but Madin-Darby canine kidney (MDCK) cells have emerged as an important alternative host. In this study, we compared glycosylation patterns, including the occupancy of potential glycosylation sites and the distribution of different glycans, on the HAs of three strains of influenza viruses for the production a trivalent seasonal flu vaccine for the 2015-2016 Northern Hemisphere season (i.e., A/California/7/2009 (H1N1) X179A, A/Switzerland/9715293/2013 (H3N2) NIB-88, and B/Brisbane/60/2008 NYMC BX-35###). Of the 8, 12, and 11 potential glycosylation sites on the HAs of H1N1, H3N2, and B strains, respectively, most were highly occupied. For the H3N2 and B strains, MDCK-derived HAs contained more sites being partially occupied (<95%) than embryo-derived HAs. A highly sensitive glycan assay was developed where 50 different glycans were identified, which was more than what has been reported previously, and their relative abundance was quantified. In general, MDCK-derived HAs contain more glycans of higher molecular weight. High-mannose species account for the most abundant group of glycans, but at a lower level as compared to those reported in previous studies, presumably due to that lower abundance, complex structure glycans were accounted for in this study. The different glycosylation patterns between MDCK- and chicken embryo-derived HAs may help elucidate the role of glycosylation on the function of influenza vaccines. KEY POINTS: • For the H3N2 and B strains, MDCK-derived HAs contained more partially (<95%) occupied glycosylation sites. • MDCK-derived HAs contained more glycans of higher molecular weight. • A systematic comparison of glycosylation on HAs used for trivalent seasonal flu vaccines was conducted.

A community outbreak of human influenza A(H1N1)pdm09 virus strains was observed in Myanmar in 2017. We investigated the circulation patterns, antigenicity, and drug resistance of 2017 influenza A(H1N1)pdm09 viruses from Myanmar and characterized the full genome of influenza virus strains in Myanmar from in-patients and out-patients to assess the pathogenicity of the viruses. Nasopharyngeal swabs were collected from out-patients and in-patients with acute respiratory tract infections in Yangon and Pyinmana City in Myanmar during January-December 2017. A total of 215 out-patients and 18 in-patients infected with A(H1N1)pdm09 were detected by virus isolation and real-time RT-PCR. Among the positive patients, 90.6% were less than 14 years old. Hemagglutination inhibition (HI) antibody titers against A(H1N1)pdm09 viruses in Myanmar were similar to the recommended Japanese influenza vaccine strain for 2017-2018 seasons (A/Singapore/GP1908/2015) and WHO recommended 2017 southern hemisphere vaccine component (A/Michigan/45/2015). Phylogenetic analysis of the hemagglutinin sequence showed that the Myanmar strains belonged to the genetic subclade 6B.1, possessing mutations of S162N and S164T at potential antigenic sites. However, the amino acid mutation at position 222, which may enhance the severity of disease and mortality, was not found. One case with no prior history of oseltamivir treatment possessed H275Y mutated virus in neuraminidase (NA), which confers resistance to oseltamivir and peramivir with elevated IC50 values. The full genome sequence of Myanmar strains showed no difference between samples from in-patients and out-patients, suggesting no additional viral mutations associated with patient severity. Several amino acid changes were observed in PB2, PB1, and M2 of Myanmar strains when compared to the vaccine strain and other Asian strains. However, no mutations associated with pathogenicity were found in the Myanmar strains, suggesting that viral factors cannot explain the underlying reasons of the massive outbreak in Myanmar. This study reported the first detection of an oseltamivir-resistant influenza virus in Myanmar, highlighting the importance of continuous antiviral monitoring and genetic characterization of the influenza virus in Myanmar.

We compared complete genome sequences of two strains of an avian influenza A (H5N6) virus isolated from a patient in Anhui Province with those of other strains from GenBank and Global initiative on sharing all influenza data (GISAID). The HA gene of the isolated virus shared homology with that of A/chicken/Zhejiang/727155/2014 (H5N6) at the level of similarity of 98%. The six internal genes of the Anhui strains were close to those of H9N2 viruses from Zhejiang, Shandong, and Guangdong provinces, with a similarity of 99%. In addition, the similarity between the internal antigens (NP and MP) of the isolated H5N6 virus and H7N9 and H10N8 viruses was 99%. Based on the data of phylogenetic analysis, the H5N6 influenza virus isolated in Anhui Province belonged to clade 2.3.4.4. The virus was shown to have molecular characteristics of highly pathogenic avian influenza viruses, including eight glycosylation sites and an amino acid sequence of the HA protein cleavage site, PLRERRRKKR/GLF, containing multiple basic amino acids. Additionally, the stalk domain of the NA protein was found to have a deletion in NA stalk region (11 amino acids in N6, positions 58-68). Our study demonstrated that the H5N6 virus from Anhui Province represented a triple-reassortant virus and could be highly pathogenic to humans. The prevalence of this virus should be closely monitored.

Long-chain multiantenna N-glycans are extremely complex molecules. Their inherent flexibility and the presence of repetitions of monosaccharide units in similar chemical environments hamper their full characterization by X-ray diffraction or standard NMR methods. Herein, the successful conformational and interaction analysis of a sialylated tetradecasaccharide N-glycan presenting two LacNAc repetitions at each arm is presented. This glycan has been identified as the receptor of the hemagglutinin protein of pathogenic influenza viruses. To accomplish this study, a N-glycan conjugated with a lanthanide binding tag has been synthesized, enabling analysis of the system by paramagnetic NMR. Under paramagnetic conditions, the NMR signals of each sugar unit in the glycan have been determined. Furthermore, a detailed binding epitope of the tetradecasaccharide N-glycan in the presence of HK/68 hemagglutinin is described.

The objective of this study was to estimate influenza vaccine effectiveness (VE) for the 2016/17 epidemic of co-circulating influenza A(H1N1)pdm09 and A(H3N2) viruses in Beijing, the capital of China.
The surveillance-based study included all swabbed patients through influenza virological surveillance, between November 2016 and April 2017. A test-negative case-control design was used to estimate influenza VE against medically-attended laboratory-confirmed influenza in outpatient settings. Cases were influenza-like illness (ILI) patients who tested positive for influenza, and controls were influenza negative patients.
A total of 10,496 ILI patients were enrolled and swabbed. Among them, 735 tested positive for influenza A(H1N1)pdm09, 1851 for A(H3N2), and 40 for type B. Of the 45 randomly selected specimens out of 1851 influenza A(H3N2) viruses, 2(4.4%) belonged to the H3N2 3C.2a1 clade, and 43(95.6%) belonged to A/Hong Kong/4801/2014-like 3C.2a clade. Among the 43 viruses of the 3C.2a clade, 32 viruses clustered in one subgroup carrying T131K, R142K and R261Q substitutions. The adjusted VE against all influenza was low at 25% (95% confidence interval (CI): 0-43%), with 54% (95%CI: 22-73%) for influenza A(H1N1)pdm09, and 2% (95%CI: -35% to 29%) for influenza A(H3N2).
Our study suggested a moderate VE against influenza A(H1N1)pdm09, but low VE against influenza A(H3N2) in Beijing, 2016/17 season. Amino acid substitutions in the hemagglutinin may contribute to the low VE against influenza A(H3N2) for this season.

H9N2 avian influenza viruses (AIVs) have been recorded in Eurasian for several years. Since 2004-2005, the disease has become endemic in Iraq, causing serious economic losses in the poultry industry. The hemagglutinin (HA) and neuraminidase (NA), two out of eight protein-coding genes, play an important role during the early stage of infection and hinder virus assembling. Little is known about the genetic information of the H9N2 viruses currently circulating in Iraq; thus, gene sequences of six AIVS of the H9N2 subtype have been detected and analyzed in the period of 2014-2015 from different outbreaks of broiler flocks in five provinces situated in the middle and southern parts of Iraq. Genetic comparison of the partial sequences of HA gene indicated that all Iraqi viruses are related to each other and could be divided into two subgroups. Viruses of the first and the second subgroups demonstrated a high similar identity with Pakistani and Iranian viruses, respectively. The nucleotide sequences of the NA protein of the all studied Iraqi viruses were very similar (95.2-100% identity), and shared high nucleotide sequence identity with Iranian, Pakistani, and Lebanese strains. All six recent viruses possessed histidine, alanine, and leucine at positions 183, 190, and 226, respectively, which are the key residues in receptor-binding sites. The Iraqi viruses were closely related to viruses of G1-like lineage isolated from poultry flocks of Iran and Pakistan, suggesting that possible epidemiological links could be derived from a common origin. Further investigations are required and should include the viral isolation and full-length molecular characterization of H9N2 AIVs in this area.

In May 2014, China formally confirmed the first human infection with the novel H5N6 avian influenza virus (AIV) in Sichuan Province. Before the first human case was reported, surveillance of AIVs in wild birds resulted in the detection of three H5N6 viruses in faecal samples from migratory waterfowl in Chenhu wetlands, Hubei Province, China. Genetic and phylogenetic analyses revealed that these three novel viruses were closely related to the H5N6 virus that has caused human infections in China since 2014. A Bayesian phylogenetic reconstruction of all eight segments suggests multiple reassortment events in the evolution of these viruses. The hemagglutinin (HA) and neuraminidase (NA) originated from the H5N2 and H6N6 AIVs, respectively, whereas all six internal genes were derived from avian H5N1 viruses. The reassortant may have occurred in eastern China during 2012-2013. A phylogeographic analysis of the HA and NA genes traced the viruses to southern China, from where they spread to other areas via eastern China. A receptor-binding test showed that H5N6 viruses from migratory waterfowl had human-type receptor-binding activity, suggesting a potential for transmission to humans. These data suggest that migratory waterfowl may play a role in the dissemination of novel H5N6 viruses.

Influenza viruses evolve at such a high rate that vaccine recommendations need to be changed, but not quite on a regular basis. This observation suggests that the rate of evolution of these viruses is not constant through time, which begs the question as to when such rate changes occur, if they do so independently of the host in which they circulate and (or) independently of their subtype. To address these outstanding questions, we introduce a novel heuristics, Mclust*, that is based on a two-tier clustering approach in a phylogenetic context to estimate (i) absolute rates of evolution and (ii) when rate change occurs. We employ the novel approach to compare the two influenza surface proteins, hemagglutinin and neuraminidase, that circulated in avian, human, and swine hosts between 1960 and 2014 in two subtypes: H3N2 and H1N1. We show that the algorithm performs well in most conditions, accounting for phylogenetic uncertainty by means of bootstrapping and scales up to analyze very large data sets. Our results show that our approach is robust to the time-dependent artifact of rate estimation, and confirm pervasive punctuated evolution across hosts and subtypes. As such, the novel approach can potentially detect when vaccine composition needs to be updated.

Oseltamivir-resistant 2009 H1N1 influenza virus infections associated with neuraminidase (NA) H275Y have been identified sporadically. Strains possessing the hemagglutinin (HA) D222G mutation have been detected in small numbers of fatal 2009 H1N1 cases. We report the first clinical description of 2009 H1N1 virus infection with both NA-H275Y and HA-D222G mutations detected by pyrosequencing of bronchioalveolar lavage fluid obtained on symptom day 19. The 59-year-old immunosuppressed patient had multiple conditions conferring higher risk of prolonged viral replication and severe illness and died on symptom day 34. Further investigations are needed to determine the significance of infection with strains possessing NA-H275Y and HA-D222G.