I am a PhD student in Medical Microbiology working on influenza virus in over 3,500 samples.
First evidence of molecular detection of influenza A virus in pig-handlers in Nigeria and Africa.
Background: Influenza A viruses in pigs have been scarcely investigated in Africa, with rare viral detection prior to 2009. The spread of pandemic H1N1/2009 changed the epidemiology of swine influenza due to frequent human-to-swine transmissions. New reassortants are emerging with combination of genes from swine influenza and pandemic H1N1/2009 strains. This underscores the need for continuous surveillance in pigs that are considered as “mixing vessels” for influenza A viruses. Also, pig-handlers faced with occupational hazards are crucial players in the inter-species transmission of influenza A viruses. This investigation was therefore intended to carry out serological and molecular surveillance of influenza A virus from pigs and pig-handlers in Lagos, Nigeria. Methods: This study was designed as a cross-sectional epidemiological surveillance of influenza A virus in pigs and pig-handlers. Between April, 2013 and June, 2014, 7 different pig farm locations were visited in Lagos. In those farms, many pig handlers rear pigs of various ages in very close proximity in open pens on the same farm land, undermining biosecurity. A total of 235 pig serum samples were analysed by competition ELISA (Idexx, France) to detect antibodies against influenza A nucleocapsid protein. Nasal and oropharyngeal swabs from pigs (n=1200) and human (n=460) obtained were investigated for the presence of influenza A virus by matrix gene RT-PCR, which were then subtyped and sequenced. Results: Findings showed that 58 (24.7%) pigs had anti-influenza A antibodies but none of the pig swabs were positive by RT-PCR. Two influenza A viruses were detected in pig-handlers and characterized as pandemic H1N1/2009 and seasonal human H3N2. Conclusions: Seroprevalence data of influenza A virus in the virtually healthy pig population category have provided evidence of sub-clinical viral circulation. Non-vaccination practice in the pig population studied indicates exposure to wild type strains. This study is the first evidence of molecular detection of influenza A virus in pig-handlers in Nigeria and Africa to the best of our knowledge. Even if the viruses identified are typical human seasonal influenza viruses and are not linked to swine husbandry, sick pig-handlers attending to animals can transmit human influenza viruses to their pigs, a phenomenon known as anthroponosis or reverse zoonosis. More awareness of pig-handlers towards reverse zoonosis is needed to avoid the generation of reassortants.
Abstract: Reassortment of gene segments between co-infecting influenza A viruses (IAV) facilitates viral diversification and has significant epidemiological impact on seasonal and pandemic influenza. Since 1977, human IAVs of H1N1 and H3N2 subtypes have co-circulated with relatively few documented cases of reassortment. We evaluated the potential for viruses of the 2009 pandemic H1N1 (pH1N1) and seasonal H3N2 lineages to reassort under experimental conditions. Results of heterologous co-infections with pH1N1 and H3N2 viruses were compared to those obtained following co-infection with homologous, genetically tagged, pH1N1 viruses as a control. High genotype diversity was observed among progeny of both co-infections; however, diversity was more limited following heterologous co-infection. Pairwise analysis of genotype patterns revealed that homologous reassortment was random while heterologous reassortment was characterized by specific biases. pH1N1/H3N2 reassortant genotypes produced under single cycle co-infection conditions showed a strong preference for homologous PB2-PA combinations and general preferences for the H3N2 NA, pH1N1 M, and the H3N2 PB2 except when paired with the pH1N1 PA or NP. Multicycle co-infection results corroborated these findings and revealed an additional preference for the H3N2 HA. Segment compatibility was further investigated by measuring chimeric polymerase activity and growth of selected reassortants in human tracheobronchial epithelial cells. In guinea pigs inoculated with a mixture of viruses, parental H3N2 viruses dominated but reassortants also infected and transmitted to cage mates. Taken together, our results indicate that strong intrinsic barriers to reassortment between seasonal H3N2 and pH1N1 viruses are few, but that the reassortants formed are attenuated relative to parental strains.IMPORTANCE The genome of IAV is relatively simple, comprising eight RNA segments, each of which typically encodes one or two proteins. Each viral protein carries out multiple functions in coordination with other viral components and the machinery of the cell. When two IAV co-infect a cell, they can exchange genes through reassortment. The resultant progeny viruses often suffer fitness defects due to sub-optimal interactions among divergent viral components. The genetic diversity generated through reassortment can facilitate the emergence of novel outbreak strains. Thus, it is important to understand the efficiency of reassortment and the factors that limit its potential. The research described here offers new tools for studying reassortment between two strains of interest, and applies those tools to viruses of the 2009 pandemic H1N1 and seasonal H3N2 lineages, which currently co-circulate in humans and therefore have the potential to give rise to novel epidemic strains.
Pub.: 24 Jun '17, Pinned: 02 Jul '17
Abstract: Human influenza viruses are rapidly evolving RNA viruses that cause short-term respiratory infections with substantial morbidity and mortality in annual epidemics. Uncovering the general principles of viral coevolution with human hosts is important for pathogen surveillance and vaccine design. Protein regions are an appropriate model for the interactions between two macromolecules, but the currently used epitope definition for the major antigen of influenza viruses, namely hemagglutinin, is very broad. Here, we combined genetic, evolutionary, antigenic, and structural information to determine the most relevant regions of the hemagglutinin of human influenza A/H3N2 viruses for interaction with human immunoglobulins. We estimated the antigenic weights of amino acid changes at individual sites from hemagglutination inhibition data using antigenic tree inference followed by spatial clustering of antigenicity-altering protein sites on the protein structure. This approach determined six relevant areas (patches) for antigenic variation that had a key role in the past antigenic evolution of the viruses. Previous transitions between successive predominating antigenic types of H3N2 viruses always included amino acid changes in either the first or second antigenic patch. Interestingly, there was only partial overlap between the antigenic patches and the patches under strong positive selection. Therefore, besides alterations of antigenicity, other interactions with the host may shape the evolution of human influenza A/H3N2 viruses.
Pub.: 15 Feb '16, Pinned: 02 Jul '17
Abstract: Despite significant advancement in vaccine and virus research, influenza continues to be a major public health concern. Each year in the United States of America, influenza viruses are responsible for seasonal epidemics resulting in over 200,000 hospitalizations and 30,000-50,000 deaths. Accurate and early diagnosis of influenza viral infections are critical for rapid initiation of antiviral therapy to reduce influenza related morbidity and mortality both during seasonal epidemics and pandemics. Several different approaches are currently available for diagnosis of influenza infections in humans. These include viral isolation in cell culture, immunofluorescence assays, nucleic acid amplification tests, immunochromatography-based rapid diagnostic tests, etc. Newer diagnostic approaches are being developed to overcome the limitations associated with some of the conventional detection methods. This review discusses diagnostic approaches currently available for detection of influenza viruses in humans.
Pub.: 15 Apr '16, Pinned: 02 Jul '17
Abstract: The influenza virus H1N1 has been prevalent all over the world for nearly a century. Many studies on its evolutionary history, substitution rate and antigenicity-associated sites have been done with small datasets. To have a complete view, we analysed 3171 full-length HA sequences from human H1N1 viruses sampled from 1918 to 2016, and discovered a new clade has formed with sequences isolated in Iran. Based on genetic distance calculations, we revealed an uneven evolutionary rate among sequences isolated in different years. We also found that the HA1 fragment of the new clade is like that of viruses that existed in the 1930s, while the HA2 fragment is closely associated with strains isolated after the 2009 pandemic. This new, "mixed" HA sequence indicates a cryptic antigenic shift event occurred, and it should draw more attention to the new clade identified from sequences from Iran.
Pub.: 01 Apr '17, Pinned: 02 Jul '17
Abstract: Since the first detection of human H3N2 influenza virus in Taiwanese pigs in 1970, infection of pigs with wholly human viruses has been known to occur in other parts of the world. These viruses, referred to as human-like H3N2 viruses, have been known to cause clinical and subclinical infections of swine populations. Due to the paucity and complete unavailability of information on transmission of influenza viruses from other species, especially humans, to swine in Nigeria and Ghana, respectively, this study was designed to investigate the presence and prevalence of a human strain of influenza A (H3N2) in swine populations at three locations in two cities within these two West African countries in January and February, 2014. Using stratified random technique, nasal swab specimens were collected from seventy-five (75) pigs at two locations in Ibadan, Nigeria and from fifty (50) pigs in Kumasi, Ghana. These specimens were tested directly by a sensitive Quantitative Solid Phase Antigen-detection Sandwich ELISA using anti-A/Brisbane/10/2007 haemagglutinin monoclonal antibody. Influenza virus A/Brisbane/10/2007 (H3N2) was detected among pigs at the three study locations, with an aggregate prevalence of 4.0% for the two locations in Ibadan, Nigeria and also 4.0% for Kumasi, Ghana. Transmission of influenza viruses from other species to swine portends serious sinister prospects for genetic reassortment and evolvement of novel viruses. We therefore recommend that further studies should be carried out to investigate the presence of other circulating human and avian influenza viruses in swine populations in West Africa and also determine the extent of genetic reassortment of strains circulating among these pigs. This would provide an early warning system for detection of novel influenza viruses, which could have pandemic potentials.
Pub.: 23 Jun '15, Pinned: 02 Jul '17
Abstract: Influenza viruses are a major pathogen of both humans and animals. Recent studies using gene-knockout mice have led to an in-depth understanding of the innate sensors that detect influenza virus infection in a variety of cell types. Signalling downstream of these sensors induces distinct sets of effector mechanisms that block virus replication and promote viral clearance by inducing innate and adaptive immune responses. In this Review, we discuss the various ways in which the innate immune system uses pattern recognition receptors to detect and respond to influenza virus infection. We consider whether the outcome of innate sensor stimulation promotes antiviral resistance or disease tolerance, and propose rational treatment strategies for the acute respiratory disease that is caused by influenza virus infection.
Pub.: 26 Apr '14, Pinned: 02 Jul '17
Abstract: Respiratory disease due to influenza virus is common in both human and swine populations around the world with multiple transmission routes capable of transmitting influenza virus, including indirect routes. The objective of this study was to evaluate the role of fomites in influenza A virus (IAV) transmission between pig populations separated by two different biosecurity settings. Thirty-five pigs were divided into four experimental groups: 10 pigs (1 replicate) were assigned to the infected group (I), 10 pigs (2 replicates of 5 pigs) were assigned to the low biosecurity sentinel group (LB), 10 pigs (2 replicates of 5 pigs) were assigned to the medium biosecurity sentinel group (MB), and 5 pigs (1 replicate) were assigned to the negative control group (NC). Eight of 10 pigs in the infected group were inoculated with IAV and 36 hours following inoculation, personnel movement events took place in order to move potentially infectious clothing and personal protective equipment (PPE) to sentinel pig rooms. Following contact with the infected group, personnel moved to the MB group after designated hygiene measures while personnel moved directly to the LB group. Nasal swabs and blood samples were collected from pigs to assess IAV infection status and fomites were sampled and tested via RRT-PCR. All experimentally inoculated pigs were infected with IAV and 11 of the 144 fomite samples collected following contact with infected pigs were low level positive for IAV genome. One replicate of each sentinel groups LB and MB became infected with IAV and all five pigs were infected over time. This study provides evidence that fomites can serve as an IAV transmission route from infected to sentinel pigs and highlights the need to focus on indirect routes as well as direct routes of transmission for IAV.
Pub.: 28 Jun '13, Pinned: 02 Jul '17
Abstract: H9N2 avian influenza viruses are endemic in poultry in Asia and the Middle East. These viruses sporadically cause dead-end infections in pigs and humans raising concerns about their potential to adapt to mammals or reassort with human or swine influenza viruses. We performed ten serial passages with an avian H9N2 virus (A/quail/Hong Kong/G1/1997) in influenza naïve pigs to assess the potential of this virus to adapt to swine. Virus replication in the entire respiratory tract and nasal virus excretion were examined after each passage and we deep sequenced viral genomic RNA of the parental and passage four H9N2 virus isolated from the nasal mucosa and lung. The parental H9N2 virus caused a productive infection in pigs with a predominant tropism for the nasal mucosa, whereas only 50% lung samples were virus-positive. In contrast, inoculation of pigs with passage four virus resulted in viral replication in the entire respiratory tract. Subsequent passages were associated with reduced virus replication in the lungs and infectious virus was no longer detectable in the upper and lower respiratory tract of inoculated pigs at passage ten. The broader tissue tropism after four passages was associated with an amino acid residue substitution at position 225, within the receptor-binding site of the hemagglutinin. We also compared the parental H9N2, passage four H9N2 and the 2009 pandemic H1N1 (pH1N1) virus in a direct contact transmission experiment. Whereas only one out of six contact pigs showed nasal virus excretion of the wild-type H9N2 for more than four days, all six contact animals shed the passage four H9N2 virus. Nevertheless, the amount of excreted virus was significantly lower when compared to that of the pH1N1, which readily transmitted and replicated in all six contact animals. Our data demonstrate that serial passaging of H9N2 virus in pigs enhances its replication and transmissibility. However, full adaptation of an avian H9N2 virus to pigs likely requires an extensive set of mutations.
Pub.: 07 Apr '17, Pinned: 02 Jul '17
Abstract: Avian H9N2 and 2009 pandemic H1N1 (pH1N1) influenza viruses can infect pigs and humans, raising the concern that H9N2:pH1N1 reassortant viruses could emerge. Such reassortants demonstrated increased replication and transmissibility in pig, but were still inefficient when compared to pH1N1. Here, we evaluated if a reassortant virus containing the hemagglutinin and neuraminidase of A/quail/Hong Kong/G1/1997 (H9N2) in the A/California/04/2009 (pH1N1) backbone could become better adapted to pigs by serial passaging. The tropism of the original H9N2:pH1N1 (P0) virus was restricted to the nasal mucosa, with no virus detected in the trachea or lungs. Nevertheless, after seven passages the H9N2:pH1N1 (P7) virus replicated in the entire respiratory tract. We also compared the transmissibility of H9N2:pH1N1 (P0), H9N2:pH1N1 (P7) and pH1N1. While only 2/6 direct-contact pigs showed nasal virus excretion of H9N2:pH1N1 (P0) ≥five days, 4/6 direct-contact animals shed the H9N2:pH1N1 (P7). Interestingly, those four animals shed virus with titers similar to those of the pH1N1, which readily transmitted to all six contact animals. The broader tissue tropism and the increased post-transmission replication after seven passages were associated with the HA-D225G substitution. Our data demonstrate that the pH1N1 internal-protein genes together with the serial passages favour H9N2 virus adaptation to pigs.
Pub.: 04 May '17, Pinned: 02 Jul '17
Abstract: Pigs and humans have shared influenza A viruses (IAV) since at least 1918, and many interspecies transmission events have been documented since that time. However, despite this interplay, relatively little is known regarding IAV circulating in swine around the world compared with the avian and human knowledge base. This gap in knowledge impedes our understanding of how viruses adapted to swine or man impacts the ecology and evolution of IAV as a whole and the true impact of swine IAV on human health. The pandemic H1N1 that emerged in 2009 underscored the need for greater surveillance and sharing of data on IAV in swine. In this paper, we review the current state of IAV in swine around the world, highlight the collaboration between international organizations and a network of laboratories engaged in human and animal IAV surveillance and research, and emphasize the need to increase information in high-priority regions. The need for global integration and rapid sharing of data and resources to fight IAV in swine and other animal species is apparent, but this effort requires grassroots support from governments, practicing veterinarians and the swine industry and, ultimately, requires significant increases in funding and infrastructure.
Pub.: 06 Apr '13, Pinned: 01 Jul '17
Abstract: We report a whole-genome analysis of 19 influenza A(H1N1)pdm09 isolates from four Ugandan hospitals between 2009 and 2011. The isolates differed from the vaccine strain A/California/07/2009 by 3 amino acid substitutions P100S, S220T AND I338V in the hemagglutinin and by 2 amino acid substitutions V106I and N248D in the neuraminidase with consistent mutations in all gene segments distinguishing isolates from the 2009/2010 and 2010/2011 seasons. Phylogenetic analysis showed low genetic evolution, with genetic distances of 0%-1.3% and 0.1%-1.6% for HA and NA genes respectively. The amino acid substitutions did not lead to antigenic differences from the reference strains. This article is protected by copyright. All rights reserved.
Pub.: 25 Jun '16, Pinned: 30 Jun '17
Abstract: Information on the timing of influenza circulation remains scarce in Tropical regions of Africa.We assessed the relationship between influenza activity and several meteorological factors (temperature, specific humidity, precipitation), and characterized the timing of influenza circulation, and its implications to vaccination strategies in Kenya.We analyzed virologically-confirmed influenza data for outpatient influenza-like illness (ILI), hospitalized for severe acute respiratory infections (SARI), and cases of severe pneumonia over the period 2007-2013. Using logistic and negative binomial regression methods, we assessed the independent association between climatic variables (lagged up to 4 weeks) and influenza activity.There were multiple influenza epidemics occurring each year and lasting a median duration of 2-4 months. On average, there were two epidemics occurring each year in most of the regions in Kenya, with the first epidemic occurring between the months of February and March and the second one between July and November. Specific humidity was independently and negatively associated with influenza activity. Combinations of low temperature (<18°C) and low specific humidity (<11g/Kg) were significantly associated with increased influenza activity.Our study broadens understanding of the relationships between seasonal influenza activity and meteorological factors in the Kenyan context. While rainfall is frequently thought to be associated with influenza circulation in the tropics, the present findings suggest low humidity is more important in Kenya. If annual vaccination were a component of a vaccination strategy in Kenya, the months of April to June are proposed as optimal for associated campaigns. This article is protected by copyright. All rights reserved.
Pub.: 22 Apr '16, Pinned: 30 Jun '17
Abstract: The triple-reassortant H1N1/2009 influenza A virus, which caused the first influenza pandemic of the 21(st) century, is generally associated with mild disease and a relatively low mortality rate comparable to that of seasonal influenza virus outbreaks. There is a growing concern about the potential for reassortment between the low-mortality H1N1/2009 and other high-mortality influenza viruses. Here, we describe and characterize a novel reassortant H1N1/2009 influenza virus, isolated from a human sample, that contained an NS gene from a highly pathogenic H5N1 virus. We evaluated the effect of the acquired NS gene on viral virulence both in vitro and in vivo and found that the novel NS-reassorted influenza virus replicated well in different cell lines and several organs of BALB/c mice without prior adaption and induced a cytokine imbalance. Therefore, there is a continued risk for further reassortment of the H1N1/2009 virus, and therefore, systematic surveillance should be enhanced to prepare for the next possible pandemic.
Pub.: 20 May '17, Pinned: 29 Jun '17
Abstract: Besides birds, pigs are another important reservoir of influenza A viruses that can be transmitted to human, as highlighted by the emergence and spread of the pandemic (H1N1) virus (pdm/09) in 2009. Surveillance in pigs is therefore necessary for public health and influenza pandemic preparedness. Nevertheless, there is a serious lack of data on influenza in Africa, especially in swine. We therefore collected serum samples from pigs in Nigeria (2009, 2012) and Cameroon (2011) in which the presence of anti-influenza A neutralizing antibodies was investigated. Our serological survey suggests that, before the 2009 pandemic, only rare swine and human H3N2 or human H1N1 infections occurred in Nigeria in swine. However, in 2011-2012, 27.4% of pigs in Nigeria and 5.6% in Cameroon had antibodies against H1N1 viruses. Higher antibody titres against pdm/09 suggested that pigs were exposed to this or a similar virus, either by multiple introductions or sustained circulation, and that reactivity against American and European swine H1N1 viruses resulted from cross-reaction.
Pub.: 30 Jan '15, Pinned: 29 Jun '17
Abstract: On April 15 and April 17, 2009, novel swine-origin influenza A (H1N1) virus (S-OIV) was identified in specimens obtained from two epidemiologically unlinked patients in the United States. The same strain of the virus was identified in Mexico, Canada, and elsewhere. We describe 642 confirmed cases of human S-OIV infection identified from the rapidly evolving U.S. outbreak.Enhanced surveillance was implemented in the United States for human infection with influenza A viruses that could not be subtyped. Specimens were sent to the Centers for Disease Control and Prevention for real-time reverse-transcriptase-polymerase-chain-reaction confirmatory testing for S-OIV.From April 15 through May 5, a total of 642 confirmed cases of S-OIV infection were identified in 41 states. The ages of patients ranged from 3 months to 81 years; 60% of patients were 18 years of age or younger. Of patients with available data, 18% had recently traveled to Mexico, and 16% were identified from school outbreaks of S-OIV infection. The most common presenting symptoms were fever (94% of patients), cough (92%), and sore throat (66%); 25% of patients had diarrhea, and 25% had vomiting. Of the 399 patients for whom hospitalization status was known, 36 (9%) required hospitalization. Of 22 hospitalized patients with available data, 12 had characteristics that conferred an increased risk of severe seasonal influenza, 11 had pneumonia, 8 required admission to an intensive care unit, 4 had respiratory failure, and 2 died. The S-OIV was determined to have a unique genome composition that had not been identified previously.A novel swine-origin influenza A virus was identified as the cause of outbreaks of febrile respiratory infection ranging from self-limited to severe illness. It is likely that the number of confirmed cases underestimates the number of cases that have occurred.
Pub.: 09 May '09, Pinned: 29 Jun '17
Abstract: WHO declared pandemic of A/H1N1 influenza in 2009 following global spread of the newly emerged strain of the virus from swine. Presently there is a dearth of data on the ecology of pandemic influenza H1N1 required for planning of intervention measures in sub Saharan Africa. Herein we report isolation of 2009 pandemic influenza A/H1N1 in an intensive mega piggery farms operation in South West Nigeria. Sentinel surveillance was carried out in a cohort of intensively reared pigs over a period of two years. Nasal swab specimens were collected at monthly interval from observed clinical cases of influenza like illness in pigs and pig handlers. Samples were analyzed by real time RT-PCR and isolation in chicken embryonated eggs. A total of 227 clinical cases of influenza like illness were observed among pigs out of which 31 (13.7%) were positive for influenza A matrix gene by real time RT-PCR. Virus isolation yielded 29 (12%) isolates out of which 18 (18%) were identified as influenza A/H1N1 by Heamaglutination Inhibition test using H1 antisera. RT-PCR positive samples were subtyped as 2009 pandemic A/H1N1 with subtype specific primers and probes. This is the first report of detection and isolation of pandemic influenza H1N1 from pigs in Nigeria. Continuous circulation of this virus in pigs may cause reassortments with seasonal influenza or mutations and substitutions in the gene that may result in the emergence of novel or pandemic influenza virus of economic and public health importance. Nigeria is considered a geographical hotspot of zoonotic diseases, which necessitate active surveillance and monitoring of emerging pandemic threats.
Pub.: 11 Dec '13, Pinned: 29 Jun '17