Pandemic Virus Strains Research Articles

Reovirus Efficiently Reassorts Genome Segments during Coinfection and Superinfection.

Reassortment, or genome segment exchange, increases diversity among viruses with segmented genomes. Previous studies on the limitations of reassortment have largely focused on parental incompatibilities that restrict generation of viable progeny. However, less is known about whether factors intrinsic to virus replication influence reassortment. Mammalian orthoreovirus (reovirus) encapsidates a segmented, double-stranded RNA (dsRNA) genome, replicates within cytoplasmic factories, and is susceptible to host antiviral responses. We sought to elucidate the influence of infection multiplicity, timing, and compartmentalized replication on reovirus reassortment in the absence of parental incompatibilities. We used an established post-PCR genotyping method to quantify reassortment frequency between wild-type and genetically barcoded type 3 reoviruses. Consistent with published findings, we found that reassortment increased with infection multiplicity until reaching a peak of efficient genome segment exchange during simultaneous coinfection. However, reassortment frequency exhibited a substantial decease with increasing time to superinfection, which strongly correlated with viral transcript abundance. We hypothesized that physical sequestration of viral transcripts within distinct virus factories or superinfection exclusion also could influence reassortment frequency during superinfection. Imaging revealed that transcripts from both wild-type and barcoded viruses frequently co-occupied factories, with superinfection time delays up to 16 h. Additionally, primary infection progressively dampened superinfecting virus transcript levels with greater time delay to superinfection. Thus, in the absence of parental incompatibilities and with short times to superinfection, reovirus reassortment proceeds efficiently and is largely unaffected by compartmentalization of replication and superinfection exclusion. However, reassortment may be limited by superinfection exclusion with greater time delays to superinfection. IMPORTANCE Reassortment, or genome segment exchange between viruses, can generate novel virus genotypes and pandemic virus strains. For viruses to reassort their genome segments, they must replicate within the same physical space by coinfecting the same host cell. Even after entry into the host cell, many viruses with segmented genomes synthesize new virus transcripts and assemble and package their genomes within cytoplasmic replication compartments. Additionally, some viruses can interfere with subsequent infection of the same host or cell. However, spatial and temporal influences on reassortment are only beginning to be explored. We found that infection multiplicity and transcript abundance are important drivers of reassortment during coinfection and superinfection, respectively, for reovirus, which has a segmented, double-stranded RNA genome. We also provide evidence that compartmentalization of transcription and packaging is unlikely to influence reassortment, but the length of time between primary and subsequent reovirus infection can alter reassortment frequency.

Molecular Epidemiology and Evolutionary Dynamics of Human Influenza Type-A Viruses in Africa: A Systematic Review.

Genomic characterization of circulating influenza type-A viruses (IAVs) directs the selection of appropriate vaccine formulations and early detection of potentially pandemic virus strains. However, longitudinal data on the genomic evolution and transmission of IAVs in Africa are scarce, limiting Africa’s benefits from potential influenza control strategies. We searched seven databases: African Journals Online, Embase, Global Health, Google Scholar, PubMed, Scopus, and Web of Science according to the PRISMA guidelines for studies that sequenced and/or genomically characterized Africa IAVs. Our review highlights the emergence and diversification of IAVs in Africa since 1993. Circulating strains continuously acquired new amino acid substitutions at the major antigenic and potential N-linked glycosylation sites in their hemagglutinin proteins, which dramatically affected vaccine protectiveness. Africa IAVs phylogenetically mixed with global strains forming strong temporal and geographical evolution structures. Phylogeographic analyses confirmed that viral migration into Africa from abroad, especially South Asia, Europe, and North America, and extensive local viral mixing sustained the genomic diversity, antigenic drift, and persistence of IAVs in Africa. However, the role of reassortment and zoonosis remains unknown. Interestingly, we observed substitutions and clades and persistent viral lineages unique to Africa. Therefore, Africa’s contribution to the global influenza ecology may be understated. Our results were geographically biased, with data from 63% (34/54) of African countries. Thus, there is a need to expand influenza surveillance across Africa and prioritize routine whole-genome sequencing and genomic analysis to detect new strains early for effective viral control.

Respiratory RNA Viruses: How to Be Prepared for an Encounter with New Pandemic Virus Strains

The characteristics of the biology of influenza viruses and coronavirus that determine the implementation of the infectious process are presented. With provision for pathogenesis of infection possible effects of serine proteinase inhibitors, heparin, and inhibitors of heparan sulfate receptors in the prevention of cell contamination by viruses are examined. It has been determined that chelators of metals of variable valency and antioxidants should be used for the reduction of replicative activity of viruses and anti-inflammatory therapy. The possibility of a pH-dependent impairment of glycosylation of cellular and viral proteins was traced for chloroquine and its derivatives. The use of low-toxicity drugs as part of adjunct therapy increases the effectiveness of synthetic antiviral drugs and interferons and ensures the safety of baseline therapy.

Are we still afraid of influenza during the Sars-Cov2 pandemic? - epidemiological analysis of influenza morbidity and vaccination in the 2019/2020 season

Influenza is an infectious respiratory disease that is caused by influenza A and B viruses in humans. Pandemic outbreaks mainly involve type A virus of zoonotic origin. The severity of the pandemic flu depends on many factors, including the virulence of the pandemic virus strain and the level of pre-existing immunity. Symptoms associated with influenza virus infection differ from mild respiratory disease limited to the upper respiratory tract and are characterized by fever, sore throat, runny nose, cough, headache, muscle pain and fatigue, as well as severe and in some cases fatal pneumonia due to influenza virus or secondary bacterial infection of the lower respiratory tract. The influenza virus is quite often subject to modifications, which makes it necessary to formulate an influenza vaccine every year to match the circulating strains. These vaccines are not mandatory and, despite the broadly understood promotion among the public, they are not widely purchased. In addition, the vaccine's effectiveness is quite low in the case of an antigenic mismatch between the vaccine and the circulating strain of the virus, which causes a still high level of illness during the year. In the 2019/2020 season, a significant decrease in the number of influenza cases was observed in Poland and in the world. In this work, we will take a closer look at the numerical values of people with influenza and discuss the possible causes of this decline. Did the Covid-19 pandemic favorably affect the incidence of another virus, such as the influenza virus, and cause its decline, or did it just overshadow the real numbers, put society to sleep, and diverted their attention?

Reverse Genetics Approaches for the Development of Influenza Vaccines.

Influenza viruses cause annual seasonal epidemics and occasional pandemics of human respiratory disease. Influenza virus infections represent a serious public health and economic problem, which are most effectively prevented through vaccination. However, influenza viruses undergo continual antigenic variation, which requires either the annual reformulation of seasonal influenza vaccines or the rapid generation of vaccines against potential pandemic virus strains. The segmented nature of influenza virus allows for the reassortment between two or more viruses within a co-infected cell, and this characteristic has also been harnessed in the laboratory to generate reassortant viruses for their use as either inactivated or live-attenuated influenza vaccines. With the implementation of plasmid-based reverse genetics techniques, it is now possible to engineer recombinant influenza viruses entirely from full-length complementary DNA copies of the viral genome by transfection of susceptible cells. These reverse genetics systems have provided investigators with novel and powerful approaches to answer important questions about the biology of influenza viruses, including the function of viral proteins, their interaction with cellular host factors and the mechanisms of influenza virus transmission and pathogenesis. In addition, reverse genetics techniques have allowed the generation of recombinant influenza viruses, providing a powerful technology to develop both inactivated and live-attenuated influenza vaccines. In this review, we will summarize the current knowledge of state-of-the-art, plasmid-based, influenza reverse genetics approaches and their implementation to provide rapid, convenient, safe and more effective influenza inactivated or live-attenuated vaccines.

A one-step duplex rRT-PCR assay for the simultaneous detection of duck hepatitis A virus genotypes 1 and 3

Duck hepatitis A virus (DHAV) is a highly infectious pathogen that causes significant bleeding lesions in the viscera of ducklings less than 3 weeks old. There are three serotypes of DHAV: serotype 1 (DHAV-1), serotype 2 (DHAV-2) and serotype 3 (DHAV-3). These serotypes have no cross-antigenicity with each other. To establish an rRT-PCR assay for the rapid detection of a mixed infection of DHAV-1 and DHAV-3, two pairs of primers and a pair of matching TaqMan probes were designed based on conserved regions of DHAV-1 VP0 and DHAV-3 VP3. Finally, we established a one-step duplex rRT-PCR assay with high specificity and sensitivity for the simultaneous detection of DHAV-1 and DHAV-3. This method showed no cross-antigenicity with the other pathogens tested, including duck plague virus, Muscovy duck parvovirus, Riemerella anatipestifer, and pathogenic E. coli from ducks. Sensitivity tests identified the minimum detection limits of this method as 98 (DHAV-1) and 10 (DHAV-3) copies/reaction. To validate the method, thirty-eight clinical samples and thirty artificially infected samples collected from dead duck embryos were studied. Thirty-seven samples were positive for DHAV-1, seventeen samples were positive for DHAV-3, and fourteen samples were positive for a mixed infection using the duplex rRT-PCR method. The method established in this study is specific, sensitive, convenient and timesaving and is a powerful tool for detecting DHAV-1, DHAV-3, and their mixed infection and for conducting surveys of pandemic virus strains.

The influenza epidemic in Russia during the 2014–2015 season

ABSTRACT The goal of this study was to compare the data on the intensity of the influenza A(H3N2) and B epidemic (especially the death toll) in the 2014–2015 season with the previous epidemic of the 2013-2014 season. The data on weekly morbidity, hospitalization, deaths from influenza, and acute respiratory diseases in different age groups of inhabitants of 59 cities located in 7 Federal districts of the Russian Federation were collected using the database of the Research Institute of Influenza. Analysis of this data showed that the influenza epidemic in 2014-2015 began earlier (in December) compared to the epidemic of 2013-2014, and spread mainly from Europe through Russia to the East. The intensity of the epidemic of 2014-2015 was higher compared to the previous one. The epidemic was more prevalent by regions and cities and a wider engagement of different age groups (except children up to 2 years of age) was observed. At the peak of the epidemic, the morbidity level was higher, the average duration of the epidemic was longer, and the number of patients among cities’ inhabitants (especially among children 7-14 years of age and adults) was higher than in the previous season. The rates of hospitalization with influenza and acute respiratory viral infections (ARVI) among patients older than 65 years were also higher (1.4 times) as well as the frequency of hospitalization with a diagnosis of “influenza” (2.7 times) and the number of deaths from laboratory confirmed influenza (1.8 times). Although the influenza pandemic virus strain A(H1N1)pdm09 was not the main causative agent of the 2015 epidemic and was distributed sporadically it still remained the leading cause of deaths from influenza in the course of this epidemic (45.5% of all cases). The deaths associated with this strain were recorded only in the European part of Russian Federation.

Superior in vitro stimulation of human CD8+ T-cells by whole virus versus split virus influenza vaccines.

Pandemic and seasonal influenza viruses cause considerable morbidity and mortality in the general human population. Protection from severe disease may result from vaccines that activate antigen-presenting DC for effective stimulation of influenza-specific memory T cells. Special attention is paid to vaccine-induced CD8+ T-cell responses, because they are mainly directed against conserved internal influenza proteins thereby presumably mediating cross-protection against circulating seasonal as well as emerging pandemic virus strains. Our study showed that influenza whole virus vaccines of major seasonal A and B strains activated DC more efficiently than those of pandemic swine-origin H1N1 and pandemic-like avian H5N1 strains. In contrast, influenza split virus vaccines had a low ability to activate DC, regardless which strain was investigated. We also observed that whole virus vaccines stimulated virus-specific CD8+ memory T cells much stronger compared to split virus counterparts, whereas both vaccine formats activated CD4+ Th cell responses similarly. Moreover, our data showed that whole virus vaccine material is delivered into the cytosolic pathway of DC for effective activation of virus-specific CD8+ T cells. We conclude that vaccines against seasonal and pandemic (-like) influenza strains that aim to stimulate cross-reacting CD8+ T cells should include whole virus rather than split virus formulations.

Reverse genetics platform construction of influenza pandemic virus strain

Reverse genetics was used to construct the platform of flu pandemic strain A/California/07/2009 (H1N1). Eight genes fragements were amplified and ligated with bidirectional vector, recombinant plasmids were co transfected to the 293 T cells and rescued the virus. Gene sequencing, antigenic analysis and growth property were used to evaluate the rescued virus. Rescued virus show the genes sequence correct, keep the same antigenicity and similar growth property compared with wild type virus. The pandemic virus reverse genetics platform of A/California/07/2009 (H1N1) were built. Based on this platform, rescued virus hold the similarity of antigenicity and growth ability with wild type virus.

Impact of influenza A(H1N1)pdm09 virus on circulation dynamics of seasonal influenza strains in Kenya.

We describe virus variations from patients with influenza-like illness before and after the appearance of influenza A(H1N1)pdm09 in Kenya during January 2008-July 2011. A total of 11,592 nasopharyngeal swabs were collected from consenting patients. Seasonal influenza B, A/H1N1, A/H3N2, A/H5N1, and influenza A(H1N1)pdm09 viruses were detected by real-time reverse transcription-polymerase chain reaction. Of patients enrolled, 2073 (17.9%) had influenza. A total of 1,524 (73.4%) of 2,073 samples were positive for influenza A virus and 549 (26.6%) were positive for influenza B virus. Influenza B virus predominated in 2008 and seasonal A(H1N1) virus predominated in the first half of 2009. Influenza A(H1N1)pdm09 virus predominated in the second half of 2009. Influenza A/H3N2 virus predominated in 2010, and co-circulation of influenza A(H1N1)pdm09 virus and influenza B virus predominated the first half of 2011. The reduction and displacement of seasonal A(H1N1) virus was the most obvious effect of the arrival of influenza A(H1N1)pdm09 virus. The decision of the World Health Organization to replace seasonal A(H1N1) virus with the pandemic virus strain for the southern hemisphere vaccine was appropriate for Kenya.

Immunobiology of Influenza Vaccines

Vaccination is the primary strategy for prevention and control of influenza. The surface hemagglutinin (HA) protein of the influenza virus contains two structural elements (head and stalk) that differ in their potential utility as vaccine targets. The head of the HA protein is the primary target of antibodies that confer protective immunity to influenza viruses. The underlying health status, age, and gene polymorphisms of vaccine recipients and, just as importantly, the extent of the antigenic match between the viruses in the vaccine and those that are circulating modulate influenza vaccine protection. Vaccine adjuvants and live attenuated influenza vaccine improve the breadth of immunity to seasonal and pandemic virus strains. Eliciting antibodies against the conserved HA stem region that cross-react with HAs within influenza virus types or subtypes would allow for the development of a universal influenza vaccine. The highly complex network of interactions generated after influenza infection and vaccination can be studied with the use of systems biology tools, such as DNA microarray chips. The use of systems vaccinology has allowed for the generation of gene expression signatures that represent key transcriptional differences between asymptomatic and symptomatic host responses to influenza infection. Additionally, the use of systems vaccinology tools have resulted in the identification of novel surrogate gene markers that are predictors of the magnitude of host responses to vaccines, which is critical to both vaccine development and public health. Identifying associations between variations in vaccine immune responses and gene polymorphisms is critical in the development of universal influenza vaccines.

Validation of the modified hemagglutination inhibition assay (mHAI), a robust and sensitive serological test for analysis of influenza virus-specific immune response

BackgroundThe hemagglutination inhibition assay (HAI) is universally regarded as the gold standard in influenza virus serology. Nevertheless, difficulties in titre readouts are common and interlaboratory variations are frequently reported. ObjectiveWe developed and validated the modified HAI to facilitate reliable, accurate and reproducible analysis of sera derived from influenza vaccination studies. Study designClinical and preclinical serum samples, NIBSC reference sera and seasonal influenza virus type A (H1N1 and H3N2) and type B antigens were employed to validate the mHAI. Moreover, pandemic virus strains (H5N1 and H1N1pdm09) were used to prove assay robustness. ResultsUtilisation of a 0.08% solution of stabilised human erythrocytes, assay buffer containing bovine serum albumin and microscopical plate readout are the major differences between the modified and standard HAI assay protocols.Validation experiments revealed that the mHAI is linear, specific and up to eightfold more sensitive than the standard HAI. In 95.6% of all measurements mHAI titres were precisely measured irrespective of the assay day, run or operator. Moreover, 96.4% (H1N1) or 95.2% (H3N2 and B), respectively, of all serum samples were determined within one dilution step of the nominal values for spiked samples. Finally, the mHAI results remained unaffected by variations in virus antigens, erythrocytes, reagents, laboratory location, sample storage conditions or matrix components. ConclusionThe modified HAI is easy to analyse, requires only a single source of erythrocytes and allows utilisation of numerous influenza virus antigens, also including virus strains which are difficult to handle by the standard HAI (e.g. H3N2, H5N1 and H1N1pdm09).

A Reassortment-Incompetent Live Attenuated Influenza Virus Vaccine for Protection against Pandemic Virus Strains

Although live-attenuated influenza vaccines (LAIV) are safe for use in protection against seasonal influenza strains, concerns regarding their potential to reassort with wild-type virus strains have been voiced. LAIVs have been demonstrated to induce enhanced mucosal and cell-mediated immunity better than inactivated vaccines while also requiring a smaller dose to achieve a protective immune response. To address the need for a reassortment-incompetent live influenza A virus vaccine, we have designed a chimeric virus that takes advantage of the fact that influenza A and B viruses do not reassort. Our novel vaccine prototype uses an attenuated influenza B virus that has been manipulated to express the ectodomain of the influenza A hemagglutinin protein, the major target for eliciting neutralizing antibodies. The hemagglutinin RNA segment is modified such that it contains influenza B packaging signals, and therefore it cannot be incorporated into a wild-type influenza A virus. We have applied our strategy to different influenza A virus subtypes and generated chimeric B/PR8 HA (H1), HK68 (H3), and VN (H5) viruses. All recombinant viruses were attenuated both in vitro and in vivo, and immunization with these recombinant viruses protected mice against lethal influenza A virus infection. Overall, our data indicate that the chimeric live-attenuated influenza B viruses expressing the modified influenza A hemagglutinin are effective LAIVs.

Pandemic H1N1 Influenza A Viruses Are Resistant to the Antiviral Activities of Innate Immune Proteins of the Collectin and Pentraxin Superfamilies

Acquired immune responses elicited to recent strains of seasonal H1N1 influenza viruses provide limited protection against emerging A(H1N1) pandemic viruses. Accordingly, pre-existing or rapidly induced innate immune defenses are of critical importance in limiting early infection. Respiratory secretions contain proteins of the innate immune system, including members of the collectin and pentraxin superfamilies. These mediate potent antiviral activity and act as an initial barrier to influenza infection. In this study, we have examined the sensitivity of H1N1 viruses, including pandemic virus strains, for their sensitivity to collectins (surfactant protein [SP]-D and mannose-binding lectin [MBL]) and to the pentraxin PTX3. Human SP-D and MBL inhibited virus-induced hemagglutinating activity, blocked the enzymatic activity of the viral neuraminidase, and neutralized the ability of H1N1 viruses to infect human respiratory epithelial cells in a manner that correlated with the degree of glycosylation in the globular head of the hemagglutinin. Recent seasonal H1N1 viruses expressed three to four N-glycosylation sequons on the head of hemagglutinin and were very sensitive to inhibition by SP-D or MBL, whereas A(H1N1) pandemic viruses expressed a single N-glycosylation sequon and were resistant to either collectin. Of interest, both seasonal and pandemic H1N1 viruses were resistant to PTX3. Thus, unlike recent seasonal H1N1 strains of influenza virus, A(H1N1) pandemic viruses are resistant to the antiviral activities of innate immune proteins of the collectin superfamily.

Public Health and Medical Responses to the 1957-58 Influenza Pandemic

As the U.S. prepares to respond this fall and winter to pandemic (H1N1) 2009, a review of the 1957-58 pandemic of Asian influenza (H2N2) could be useful for planning purposes because of the many similarities between the 2 pandemics. Using historical surveillance reports, published literature, and media coverage, this article provides an overview of the epidemiology of and response to the 1957-58 influenza pandemic in the U.S., during which an estimated 25% of the population became infected with the new pandemic virus strain. While it cannot be predicted with absolute certainty how the H1N1 pandemic might play out in the U.S. this fall, lessons from the 1957-58 influenza pandemic provide useful and practical insights for current planning and response efforts.

Current status and progress of prepandemic and pandemic influenza vaccine development

H5N1 viruses are widely considered to be a probable cause of the next influenza pandemic. Influenza vaccines are considered to form the main prophylactic measure against pandemic influenza. The world’s population is expected to have no pre-existing immunity against the pandemic virus strain and will need two vaccine doses to acquire protective immunity. A pandemic outbreak will spread much faster than it will take for pandemic vaccines to be produced and distributed. Therefore, increasing efforts are being made to develop prepandemic vaccines that can induce broad cross-protective responses and that can be administered as soon as a pandemic is declared or even before, in order to successfully prime the immune system and allow for a rapid and protective antibody response with one dose of the pandemic vaccine. Several vaccine manufacturers have developed candidate pandemic and prepandemic vaccines, predominantly based on reverse-genetics reference strains and have improved the immunogenicity by formulating these vaccines with different adjuvants. Clinical studies with inactivated split-virion or whole-virion vaccines based on H5N1 indicate that two immunizations appear necessary to elicit the level of immunity required to meet licensure criteria. A detailed overview is given of the most successful candidate vaccines developed by seven vaccine manufacturers.

Avian Influenza H5N1: Risks at the Human–Animal Interface

Great concern has arisen over the continued infection of humans with highly pathogenic avian influenza (HPAI) of the H5N1 subtype. Ongoing human exposure potentially increases the risk that a pandemic virus strain will emerge that is easily transmissible among humans. Although the pathogenicity of a pandemic strain cannot be predicted, the high mortality seen in documented H5N1 human infections thus far has raised the level of concern. To define the three types of influenza that can affect humans, discuss potential exposure risks at the human-animal interface, and suggest ways to reduce exposure and help prevent development of a pandemic virus. This review is based on data and guidelines available from the World Health Organization, the scientific literature, and official governmental reports. Epidemiological data on human exposure risk are generally incomplete. Transmission of HPAI to humans is thought to occur through contact with respiratory secretions, feces, contaminated feathers, organs, and blood from live or dead infected birds and possibly from contaminated surfaces. Consumption of properly cooked poultry and eggs is not thought to pose a risk. Use of antiviral containment and vaccination may protect against development of a pandemic. To most effectively decrease the risk of a pandemic, the public health and animal health sectors--those which are responsible for protecting and improving the health of humans and animals, respectively--must collaborate to decrease human exposure to HPAI virus, both by controlling virus circulation among poultry and by assessing the risks of human exposure to avian influenza virus at the human-animal interface from primary production through consumption of poultry and poultry products, and implementing risk-based mitigation measures.