Influenza A(H1N1)pdm09 infection and viral load analysis in patients with different clinical presentations

BACKGROUND Influenza viral load (VL) can be a decisive factor in determining the antiviral efficacy in viral clearance. OBJECTIVE This study aimed to evaluate the rate of infection and the role of influenza VL on the clinical spectrum of illnesses among different patient groups attended at a tertiary hospital in Brazil. METHODS Samples were collected from patients presenting acute respiratory infection from 2009 to 2013. Overall, 2262 samples were analysed and distributed into three groups: (i) asymptomatic (AS); (ii) symptomatic outpatients (OP); and (iii) hospitalised patients (HP). VL (expressed in Log10 RNA copies/mL) was calculated through a quantitative real-time one-step reverse transcription-polymerase chain reaction (RT-PCR) assay aimed at the M gene, with human RNAseP target as internal control and normalising gene of threshold cycle values. FINDINGS A total of 162 (7.16%) H1N1pdm09 positive samples were analysed. Patients aged from 0.08 to 77 years old [median ± standard deviation (SD): 12.5 ± 20.54]. Children with 5 to 11 years old presented the highest detection (p < 0.0001). AS patients had the lowest VL, with a significant difference when compared with symptomatic patients (p = 0.0003). A higher VL was observed within two days of disease onset. Ten patients (HP group) received antiviral treatment and were followed up and presented a mean initial VL of 6.64 ± 1.82. A complete viral clearance for 50% of these patients was reached after 12 days of treatment. MAIN CONCLUSIONS It is important to evaluate AS patients as potential spreaders, as viral shedding was still present, even at lower VL. Our results suggest that patients with underlying diseases and severe clinical symptoms may be considered for prolonged viral treatment.

Molecular characterization of influenza A(H1N1)pdm09 viruses circulating at various geographical locations in India, 2017

Background & objectives: Influenza virological surveillance is an essential tool for the early detection of novel genetic variants of epidemiologic and clinical significance. This study was aimed to genetically characterize A(H1N1)pdm09 virus circulating in 2017 and to compare it with the global data. Methods: The regional/State Viral Research and Diagnostic Laboratories (VRDLs) provided influenza diagnosis for referred clinical samples and shared influenza A(H1N1)pdm09 positives with the Indian Council of Medical Research-National Institute of Virology (ICMR-NIV), Pune, India, for hemagglutinin (HA) gene phylogenetic analysis. Sites at Manipal, Jaipur and Dibrugarh performed the sequencing and shared the sequence data for analysis. The antiviral susceptibility of influenza viruses was assessed for known molecular marker H275Y at the ICMR-NIV, Pune. Results: All the eight VRDLs had well-established influenza diagnostic facilities and showed increased activity of influenza A(H1N1)pdm09 during 2017. Phylogenetic analysis showed that the viruses from the different regions of the country were similar to A/Michigan/45/2015 strain which was the 2017-2018 recommended vaccine strain and were clustered with the globally circulating clade 6B.1 with signature mutations S84N, S162N and I216T. The clade 6B.1 showed further subgrouping with additional mutations S74R, S164T and I295V; however, there was no significant association between the presence of these mutations and severity of disease due to influenza. All the study viruses were sensitive to oseltamivir. Interpretation & conclusions: During the study period, all the study sites reported globally circulating A/Michigan/45/2015 vaccine strain of influenza A(H1N1)pdm09 viruses and remained sensitive to oseltamivir. Further genetic and antigenic characterization of influenza viruses is recommended to address public health concerns.

Genetic characterisation of influenza A(H1N1)pdm09 viruses circulating in Assam, Northeast India during 2009�15

Introduction: Influenza A(H1N1)pdm09 virus, since its identification in April 2009, has continued to cause significant outbreaks of respiratory tract infections including pandemics in humans. In the course of its evolution, the virus has acquired many mutations with an ability to cause increased disease severity. A regular molecular surveillance of the virus is essential to mark the evolutionary changes that may cause a shift to the viral behavior. Materials and Methods: Samples of Throat/Nasal swabs were collected from a total of 3715 influenza-like illness cases and screened by Real-time Reverse Transcription-Polymerase Chain Reaction for influenza viruses. Nucleotide sequence analysis was done to identify changes in antigenicity of the virus strains. Results: The present study describes the molecular characteristics of influenza A(H1N1)pdm09 viruses detected in Assam of Northeast India during 2009�15. Influenza A viruses were detected in 11.4% (425/3715), of which influenza A(H1N1)pdm09 viruses were detected in 41.4% (176/425). The nucleotide sequencing of influenza A(H1N1)pdm09 viruses revealed a total of 17 and 22 amino acid substitutions in haemagglutinin (HA) and neuraminidase (NA) genes of the virus, respectively, compared to contemporary vaccine strain A/California/07/2009. The important mutations detected in HA genes of A/Assam(H1N1)pdm09 strains included E391K, K180Q and S202T. Mutation 'N248D' which has an ability to develop oseltamivir resistance was also detected in NA gene of A/Assam(H1N1)pdm09 strains. Conclusions: Regular molecular surveillance of influenza A(H1N1)pdm09 is important to monitor the viral behavior in terms of increase virulence, drug resistance pattern and emergence of novel strains.

Analysis of genomic characteristics of influenza A (H1N1) pdm09 viruses in Inner Mongolia, 2013-2017 / 中华实验和临床病毒学杂志

Objective@#To evaluate the effect of vaccine and provide scientific evidence for prevention and control of influenza virus, this study aims to analyze the characteristics of genomic variation of influenza A (H1N1) pdm09 viruses in Inner Mongolia.@*Methods@#The 16 viral strains were selected randomly according to the influenza A (H1N1) pdm09 viruses isolated from network laboratories in Inner Mongolia, 2013-2017. The hemagglutinin(HA) and neuraminidase(NA) genomic sequences were obtained by using RT-PCR and sequencing, and genomic characteristics were analyzed via bioinformatics.@*Results@#Compared to the A/California/07/2009 vaccine strain, the relatively obvious variation of antigen of influenza A (H1N1) pdm09 viruses in Inner Mongolia since 2014, and the vaccine provided a poor protection to influenza A (H1N1) pdm09 virus infection, while the A/Michigan/45/2015 vaccine strain recommended by WHO recently has a satisfactory protective effects. Several viral isolates from Inner Mongolia increased the binding force of virus in human upper respiratory tract because of D222N and D222G substitution within HA. E119K and H275Y substitution within NA gene of viral strains, suggesting that the viruses were resistant to NA inhibitors.@*Conclusions@#The influenza A (H1N1) pdm09 viruses had gradual variations as time went on, and the WHO recommended vaccine was relatively lagging. Virulent strains and drug-resistant strains appeared in the population, and the genetic characteristics of influenza virus surveillance should be strengthened to find the new mutants of virus in time, which provide evidence for the prevention and control of influenza.

Antigenic and genetic characteristics of influenza A(H1N1)pdm09 virus isolated in Sichuan province from 2018 to 2019 / 中华实验和临床病毒学杂志

Objective@#To understand the epidemiological characteristics of influenza A(H1N1)pdm09 virus in Sichuan population during the monitoring period of 2018-2019, and to clarify the antigenic variation, the gene characteristics and the matching of current epidemic strains, vaccine strains, representative strains at home and abroad.@*Methods@#A total of 118 strains of influenza A(H1N1)pdm09 virus isolated in Sichuan region influenza network laboratory from April 2018 to March 2019 were selected. The hemagglutination inhibition (HI) assay was conducted for antigen analysis. The HA and NA genes of 16 strains with low-response strains were sequenced. Phylogenetic analysis and amino acid locus variation analysis were applied using BioEdit and MEGA5.0 software.@*Results@#The result of the antigen analysis demonstrated that more than 95% of the A(H1N1) pdm09 influenza viruses detected were similar to the WHO recommended vaccine strain A/Michigan/45/2015. The analysis of HA gene characteristics showed that some low-response strains had amino acid site variation in the Sa, Sb and Cb regions of the HA protein. A total of 15 low-response strains belonged to the 6B.1 branch. And their evolutionary relationship were close to the representative strains A/beijin-xicheng/SWL1633/2018 and A/brisbane/02/2018, which were popular at home and abroad. Compared with A/sichuan/1/2009, there are mutations involving 6, 14 and 1 amino acid sites in the antigen-determining regions (Sa, Sb, Ca and Cb), non-determined regions and receptor binding site(RBS) respectively. No amino acid site mutations related to resistance to NA gene were found.@*Conclusions@#In 2018-2019, the epidemic A(H1N1) pdm09 influenza virus in Sichuan is consistent with the global epidemic characteristics, which also matched with vaccine strains recommended by WHO in the northern hemisphere. Compared with A/sichuan/1/2009 which was the first isolated in China in 2009, there were amino acid sites mutations in antigen-determining region and receptor binding site of the HA protein, and the transmembrane region of the NA protein, drug and antibody binding sites.

Complete genome analysis of influenza A(H1N1) pdm09 virus isolated from one case of pneumonia of unknown etiology (PUE) in Fujian Province, China / 中国人兽共患病学报

To study the biological characteristics and mutations of influenza A(H1N1)pdm09 virus isolated from one case of pneumonia of unknown etiology (PUE),which would provide references for clinical treatment and disease control,the throat swab specimen from the PUE case was isolated in the Madin-Darby Canine Kidney (MDCK) cells,and then the antigenicity,pathogenicity and drug resistance of influenza A (H1N1) pdm09 virus were analyzed after sequencing.As a result,one influenza virus strain was isolated from the specimen and named as A/FujianGulou/SWL64/2016(H1N1).The similarities of nucleotide sequences and amino acids sequences compared with the vaccine strain A/California/07/2009 (H1N1) were 96.9％-98.9％ and 96.7％-99.5％,respectively.Eighteen amino acids had mutated in the HA and 4 mutations,K163Q,S185T,S203T and D222N,were involved in 3 different epitopes,which indicated that the antigenic drift had occurred in the influenza virus.The D222N mutation associated with receptor binding site made the virus infect lower respiratory tract more easily.The virus was still amantadine-resistance and oseltamivir-sensitive.In conclusion,the influenza A (H1N1) pdm09 virus in this study have occurred antigenic drift and has the molecular characterization of causing severe pneumonia,so further surveillance should be performed to prevent and control the influenza epidemic.

Analysis of HA1 gene of influenza A H1N1 pdm09 virus from a clustered human cases / 中华实验和临床病毒学杂志

Objective@#To understand the viral etiology of a clustered case of human infection outbreak in the middle school of Huai’an city.@*Methods@#Nasopharyngeal swab samples from patients were collected and rapidly detected by Real-time RT-PCR and the target virus isolated in cells. Furthermore, HA1 segments of target virus were amplified by RT-PCR and sequenced. The genetic and phylogenetic analysis based on HA1 genes was computed.@*Results@#Influenza A(H1N1)pdm09 viral nucleic acid in 11 nasopharyngeal swab samples from patients in the outbreak were positive. Compared to the vaccine strains A/California/07/2009, the Huai’an isolates, nucleotide identity was 97.7%-98.1%, and amino acid identity was 96.6%-97.4%. Phylogenetic analysis of HA1 segment sequences indicated that the Huai’an strains from the outbreak were related closely to the viruses isolated in the year of 2014. Sequence analysis indicated that the Huai’an isolates had no amino acid substitution in the receptor binding sites and glycosylation sites, while in the Ca1 of antigenic determinant of HA1 the Huai’an isolates had an amino acid substitution of S for T at 220.@*Conclusions@#The pathogen of the clustered case of human infection was Influenza A(H1N1)pdm09 virus. Though the Huai’an isolates had one animo acid substitution in the Ca1 of antigenic determinant, the antigenicity characteristic remained unchanged.

Influenza A(H1N1)pdm09 cases in sub‑Himalayan region, 2014–2015 India.

Current influenza A(H1N1)pdm09 strain severely involved many parts of the country. The study was conducted to analyze the clinicoepidemiological trend of influenza A(H1N1)pdm09 cases from October 2014 to March 2015. Samples processing was done as per the Center for Disease Control guidelines. A total of 333 specimens were processed out of which influenza A(H1N1)pdm09 constituted 24% (81) of total, 5% (18) cases were seasonal influenza A virus strains. Mean age group involved was 49 years with case fatality rate of 20%. Patients died were 63% males and 44% had comorbidities, and among them, 38% patients died within 24 h of hospitalization. The mean age of comorbid patients who died was 59 years; whereas the mean age of patients died having no co‑morbidities was 41 years (P < 0.005). Mortality was seen among 81% (13) of patients who were on ventilator support. Added mortality in specific human group demands continuous surveillance monitoring followed by the detection of mutation, even in susceptible animal population.

Spatiotemporal rrends of cases of pandemic influenza a(h1n1)pdm09 in Argentina, 2009-2012 / Tendencias espacio-temporales en los casos de gripe A(H1N1)pdm09 en Argentina, 2009-2012

The aim of this paper was to analyze the spatiotemporal variations of cases of influenza A(H1N1)pdm09 in Argentina. A space-time permutation scan statistic was performed to test the non-randomness in the interaction between space and time in reported influenza A(H1N1)pdm09 cases. In 2009, two clusters were recorded in the east of Buenos Aires Province (May and June) and in the central and northern part of Argentina (July and August). Between 2011 and 2012, clusters near areas bordering other countries were registered. Within the clusters, in 2009, the high notification rates were first observed in the school-age population and then extended to the older population (15-59 years). From 2011 onwards, higher rates of reported cases of influenza A(H1N1)pdm09 occurred in children under five years in center of the country. Two stages of transmission of influenza A(H1N1)pdm09 can be characterized. The first stage had high rates of notification and a possible interaction with individuals from other countries in the major cities of Argentina (pattern of hierarchy), and the second stage had an increased interaction in some border areas without a clear pattern of hierarchy. These results suggest the need for greater coordination in the Southern Cone countries, in order to implement joint prevention and vaccination policies.

El objetivo de este trabajo es analizar las variaciones espaciotemporales de los casos de gripe A(H1N1)pdm09 en Argentina. Se realizó un escaneo estadístico espacio-temporal por permutaciones para poner a prueba la no aleatoriedad en la interacción entre espacio y tiempo de los casos registrados de gripe A(H1N1)pdm09. Durante 2009 se identificaron dos conglomerados espacio-temporales, en el este de la provincia de Buenos Aires (mayo y junio) y en la mayor parte del centro-norte de la Argentina (julio y agosto). Durante 2011 y 2012 se registraron conglomerados próximos a zonas limítrofes con otros países. Al interior de los conglomerados, primero se observaron mayores tasas de notificación en población de edad escolar para luego extenderse a población mayor (15-59 años). A partir de 2011, las mayores tasas se observaron en menores de 5 años residentes en el centro del país. Se pudieron caracterizar dos etapas de transmisión espacio-temporal de la gripe A(H1N1)pdm09. La primera etapa se caracterizó por altas tasas de notificación y una posible interacción con individuos provenientes de otros países llegados a las grandes ciudades de la Argentina (patrón de jerarquía). La segunda etapa mostró una mayor interacción en algunas zonas fronterizas y sin un patrón claro de jerarquía. Estos resultados plantean la necesidad de generar una mayor coordinación en países del Cono Sur, con el objetivo de implementar políticas más efectivas de prevención y vacunación.

Oseltamivir-resistant influenza A(H1N1)pdm2009 strains found in Brazil are endowed with permissive mutations, which compensate the loss of fitness imposed by antiviral resistance

The 2009 pandemic influenza A virus outbreak led to the systematic use of the neuraminidase (NA) inhibitor oseltamivir (OST). Consequently, OST-resistant strains, carrying the mutation H275Y, emerged in the years after the pandemics, with a prevalence of 1-2%. Currently, OST-resistant strains have been found in community settings, in untreated individuals. To spread in community settings, H275Y mutants must contain additional mutations, collectively called permissive mutations. We display the permissive mutations in NA of OST-resistant A(H1N1)pdm09 virus found in Brazilian community settings. The NAs from 2013 are phylogenetically distinct from those of 2012, indicating a tendency of positive selection of NAs with better fitness. Some previously predicted permissive mutations, such as V241I and N369K, found in different countries, were also detected in Brazil. Importantly, the change D344N, also predicted to compensate loss of fitness imposed by H275Y mutation, was found in Brazil, but not in other countries in 2013. Our results reinforce the notion that OST-resistant A(H1N1)pdm09 strains with compensatory mutations may arise in an independent fashion, with samples being identified in different states of Brazil and in different countries. Systematic circulation of these viral strains may jeopardise the use of the first line of anti-influenza drugs in the future. (AU)

Molecular findings from influenza A(H1N1)pdm09 detected in patients from a Brazilian equatorial region during the pandemic period

After the World Health Organization officially declared the end of the first pandemic of the XXI century in August 2010, the influenza A(H1N1)pdm09 virus has been disseminated in the human population. In spite of its sustained circulation, very little on phylogenetic data or oseltamivir (OST) resistance is available for the virus in equatorial regions of South America. In order to shed more light on this topic, we analysed the haemagglutinin (HA) and neuraminidase (NA) genes of influenza A(H1N1)pdm09 positive samples collected during the pandemic period in the Pernambuco (PE), a northeastern Brazilian state. Complete HA sequences were compared and amino acid changes were related to clinical outcome. In addition, the H275Y substitution in NA, associated with OST resistance, was investigated by pyrosequencing. Samples from PE were grouped in phylogenetic clades 6 and 7, being clustered together with sequences from South and Southeast Brazil. The D222N/G HA gene mutation, associated with severity, was found in one deceased patient that was pregnant. Additionally, the HA mutation K308E, which appeared in Brazil in 2010 and was only detected worldwide the following year, was identified in samples from hospitalised cases. The resistance marker H275Y was not identified in samples tested. However, broader studies are needed to establish the real frequency of resistance in this Brazilian region.

Clinical profile of pneumonia and its association with rain wetting in patients admitted at a tertiary care institute during pandemic of influenza A (H1N1) pdm09 virus infection.

Background. Influenza pneumonia often occurs as epidemics in the Asian countries and have significant impact on the health of world population. Methods. We studied the association of rain-wetting with occurrence of pneumonia during the outbreak of the influenza A (H1N1) pdm09 virus infection. All patients admitted with community-acquired pneumonia during the period 13th September to 10th October 2010 were recruited in the present study. The diagnosis of influenza was established by realtime polymerase chain reaction (RT-PCR). The demographic data and clinical profile of the patients were recorded with a special consideration to record of possible risk factors. Results. Of the 123 patients studied, 39 (32%) patients had tested positive for influenza A (H1N1) pdm09; 12 (10%) tested positive for influenza A and remaining 72 (58%) patients were negative for influenza virus. Pattern of illness was almost identical in H1N1-positive and-negative groups. History of rain-wetting was present in 48 patients (39%) preceding the onset of illness. Getting wet in the rain was significantly higher in patients with pneumonia than control subjects [odds ratio 2.53, 95% confidence intervals (CI) 1.301 - 4.91; p=0.009)]. The number of pneumonia patients was also higher on rainy days and the numbers started declining a week later. Conclusion. More pneumonia patients are admitted during the periods of greater rainfall and rain-wetting may be an important risk factor for the occurrence of pneumonia.