Large-Scale Phylogenetic Analysis of Emerging Infectious Diseases

To the Editor: In February 2013, human infection with reassortant avian influenza A(H7N9) virus occurred in eastern China.A total of 135 laboratory-confirmed cases and 44 deaths among case-patients have been reported as of August 11, 2013.Unlike infection with other H7 subtype viruses (e.g., H7N2, H7N3, and H7N7), which often cause mild-tomoderate-human disease (1), infection with H7N9 subtype virus caused severe pneumonia and acute respiratory distress syndrome in most laboratory-confirmed case-patients (2,3).Pregnant women are particularly susceptible to severe complications from influenza (seasonal and pandemic), and have an increased risk for maternal death (4).On March 30, 2013, a 25-yearold pregnant woman came to the outpatient department of a hospital in Zhenjiang, Jiangsu Province, China.She had cough and fever (temperature 38.0°C), which had begun 2 days earlier.She also reported mild myalgia and mild sore throat.The patient had no any underlying medical conditions and

Public health administrators do not have effective models to predict excess influenza-associated mortality and monitor viral changes associated with it. This study evaluated the effect of matching/mismatching vaccine strains, type/subtype pattern changes in Taiwan's influenza viruses, and the impact of post-SARS (severe acute respiratory syndrome) public health efforts on excess influenza-associated mortalities among the elderly. A negative binomial model was developed to estimate Taiwan's monthly influenza-associated mortality among the elderly. We calculated three winter and annual excess influenza-associated mortalities [pneumonia and influenza (P&I), respiratory and circulatory, and all-cause] from the 1999–2000 through the 2006–2007 influenza seasons. Obtaining influenza virus sequences from the months/years in which death from P&I was excessive, we investigated molecular variation in vaccine-mismatched influenza viruses by comparing hemagglutinin 1 (HA1) of the circulating and vaccine strains. We found that the higher the isolation rate of A (H3N2) and vaccine-mismatched influenza viruses, the greater the monthly P&I mortality. However, this significant positive association became negative for higher matching of A (H3N2) and public health efforts with post-SARS effect. Mean excess P&I mortality for winters was significantly higher before 2003 than after that year [mean ± S.D.: 1.44±1.35 vs. 0.35±1.13, p = 0.04]. Further analysis revealed that vaccine-matched circulating influenza A viruses were significantly associated with lower excess P&I mortality during post-SARS winters (i.e., 2005–2007) than during pre-SARS winters [0.03±0.06 vs. 1.57±1.27, p = 0.01]. Stratification of these vaccine-matching and post-SARS effect showed substantial trends toward lower elderly excess P&I mortalities in winters with either mismatching vaccines during the post-SARS period or matching vaccines during the pre-SARS period. Importantly, all three excess mortalities were at their highest in May, 2003, when inter-hospital nosocomial infections were peaking. Furthermore, vaccine-mismatched H3N2 viruses circulating in the years with high excess P&I mortality exhibited both a lower amino acid identity percentage of HA1 between vaccine and circulating strains and a higher numbers of variations at epitope B. Our model can help future decision makers to estimate excess P&I mortality effectively, select and test virus strains for antigenic variation, and evaluate public health strategy effectiveness.

Distinguishing the novel coronavirus from influenza

The zoonotic transmission of influenza A viruses (IAVs) and coronaviruses (CoVs) may result in severe disease. Cleavage of the surface glycoproteins hemagglutinin (HA) and spike protein (S), respectively, is essential for viral infectivity. The transmembrane serine protease 2 (TMPRSS2) is crucial for cleaving IAV HAs containing monobasic cleavage sites and severe acute respiratory syndrome (SARS)-CoV-2 S in human airway cells. Here, we analysed and compared the TMPRSS2-dependency of SARS-CoV, Middle East respiratory syndrome (MERS)-CoV, the 1918 pandemic H1N1 IAV and IAV H12, H13 and H17 subtypes in human airway cells. We used the peptide-conjugated morpholino oligomer (PPMO) T-ex5 to knockdown the expression of active TMPRSS2 and determine the impact on virus activation and replication in Calu-3 cells. The activation of H1N1/1918 and H13 relied on TMPRSS2, whereas recombinant IAVs carrying H12 or H17 were not affected by TMPRSS2 knockdown. MERS-CoV replication was strongly suppressed in T-ex5 treated cells, while SARS-CoV was less dependent on TMPRSS2. Our data underline the importance of TMPRSS2 for certain (potentially) pandemic respiratory viruses, including H1N1/1918 and MERS-CoV, in human airways, further suggesting a promising drug target. However, our findings also highlight that IAVs and CoVs differ in TMPRSS2 dependency and that other proteases are involved in virus activation.

Objective To analyze epidemiological and genetic characteristics of the fifth avian influenza A (H7N9) wave in Suzhou, and to provide scientific basis for prevention and control of H7N9 virus infection. Methods Respectively, influenza A/B, H1N1 (pdm09), H3, H5N1, and H7N9 real-time PCR kits were used to detected pharyngeal swab samples which were collected from severe acute respiratory syndrome infection (SARI) cases in Suzhou. The H7N9-positive samples were further examined for virus isolation and gene sequencing. Results The H7N9 virus was mainly prevalent in winter in Suzhou City. In the fifth H7N9 virus epidemic, the overall fatality rate of human infection with H7N9 virus in Suzhou city was 40% (22/55). Additionally, most were older people (median age was 58 years) and more than 80% of H7N9 patients had live poultry exposure history. The nucleic acid homology of HA gene was 98.7-100%. There were no mutations in the key sites of the HA gene sequence. Conclusions The H7N9 virus can not be effectively spread in the crowd currently, with no significant changes in receptor binding sites (RBS). In addition, exposure to live poultry or contaminated environment is still the main source of human infection with H7N9. At present, the viruses circulating in Suzhou city are low pathogenic to poultry. Key words: Avian influenza; Hamagglutinin gene; Severe acute respiratory syndrome infection (SARI); Real-time PCR; Influenza A (H7N9)

Viral replication is dependent on a host; with their small genomes, viruses need to hijack host cellular machinery to complete their life cycle. The coevolution of intimate virus-host relationships has led to many viruses being able to successfully propagate without a significant detrimental effect to the host. However, in some instances, viral infection causes disease. The molecular mechanisms underlying virus-associated disease were the focus of an Advanced Summer School in Africa held in Hermanus, South Africa, from 6 to 14 March 2010. Assembled in the coastal town of Hermanus were 45 participants, primarily PhD students and postdocs from less developed countries, and 18 speakers, leaders in their respective fields, from around the world ( Fig. 1). The school, the second of its kind in Africa was sponsored by the International Union of Biochemistry and Molecular Biology (IUBMB), the Federation of European Biochemical Societies (FEBS), the Federation of African Societies of Biochemistry and Molecular Biology, the United Nations Educational Scientific and Cultural Organization, and the International Centre for Genetic Engineering and Biotechnology (ICGEB). The course was honored by the presence of Brian Clark, Professor of Biostructural Chemistry at The University of Aarhus, Denmark. Prof Clark is a renowned scientist who participated in the discovery of the initiation codon for protein synthesis (1-4) and the first crystallization of a tRNA (5). Today, Prof Clark studies the molecular and cellular mechanisms of ageing using a systems biology approach. His presence was particularly significant at the Summer School since he has been involved in the organization of several similar events during his tenure as President of the IUBMB and FEBS, most notably the Spetses Summer School. Prof Clark stressed the importance of such meetings for the development, not just of young scientists, but of science itself. Participants of the Second Advancement Summer School in Africa. 45 young researchers and 18 speakers were assembled in the beautiful coastal town of Hermanus, South Africa. The main themes of discussion at the summer school were: 1) why viral infection can lead to cancer; 2) how a greater understanding of the mechanisms underpinning human immunodeficiency virus (HIV) propagation can inform new antiviral strategies; 3) the abilities of viruses to evade the immune system and the obstacles to the development of effective vaccines; and, 4) the potential afforded by viruses as research tools. The importance of host factors became apparent in the discussion of all these topics, and how viral research has informed our general knowledge of cell biology. This report serves to summarise the findings presented at the summer school. Until Peyton Rous discovered the first tumor virus in 1911, viruses were viewed as peculiar infectious agents capable of inducing cancer in animals, but not in humans. However, it is now well-established that many different human viruses, including Human papilloma virus (HPV), Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpes virus (KSHV), Human T-cell leukemia virus-1 (HTLV-1), Hepatitis B virus (HBV), Hepatitis C virus (HCV), and Merkel cell polyoma virus (MCV) are the etiological agents of human cancers, encompassing at least 15–20% of all tumors worldwide (6). Hence, tumor virology was a focus of this summer school. Dr Lawrence Banks from the ICGEB, Trieste, provided an overview of HPV and the role of viral proteins in the transformation of cervical epithelial cells. Almost all (99.7%) cervical cancers contain HPV DNA, usually types 16 and 18 (7). Despite this statistic, cancer induction is not part of the 'normal' HPV life cycle. Only in rare cases where there has been a lack of immune clearance leading to persistent infection, along with additional changes in the cell, does cancer develop. Banks described how HPV E6 and E7 are the major viral oncoproteins involved in the development of cervical cancer. The HPV life cycle is critically dependent on the differentiation of epithelial cells: there is usually no DNA replication, other than in cells of the basement layer, but HPV E6 and E7 induce a pseudo-S phase, creating a permissive environment for viral replication (8). In addition, E6 and E7 target cellular substrates for proteasome-mediated degradation, notably the tumor suppressors p53 and pRb, respectively (9, 10). HPV DNA exists in an episomal form but may be found integrated in malignant cells. Integration often involves loss of viral DNA. Indeed, malignant cells often display no viral replication but maintain expression of E6 and E7 (11), further emphasizing the roles of these proteins in cancer progression. But why do only a few HPV infections result in malignancy? This issue was tackled by Dr John Doorbar, National Institute for Medical Research, London. HPV-associated cancers are more predominant in some tissues than others: there are ∼500,000 cases of cervical cancer reported each year. In contrast, HPV-associated penile cancers are rare (∼40,000 cases per year), despite no evidence of a disparity in primary infection rates. This implies a difference in the propensity of HPV to form neoplastic lesions at different sites. Doorbar speculates that this is due to a graded ability of HPV to replicate in different tissues. As further evidence, HPV-1 causes warts but no lesions at mucosal sites. Doorbar believes that the transformation zone in the cervix may be particularly susceptible to neoplasia formation. The exact host factors responsible for the variation in HPV replication ability are unresolved. Doorbar's group is also investigating the molecular basis of the different outcomes resulting from HPV infection. HPV gene expression occurs in an ordered manner as the infected cells move through the epithelial tissue. Doorbar described how the host protein mini chromosome maintenance protein 7 (MCM7) could be used as a marker for cell proliferation and, thus, as a surrogate for E6/7 activity. MCM7 was expressed in the lower layers of HPV lesions, as shown by laser capture microscopy, and the viral protein E4 was abundantly expressed in the upper layers (12). Interestingly, Doorbar's group have shown that high-grade squamous intraepithelial lesions (HSILs) are associated with increased MCM7 expression in upper epithelial layers with almost no E4 expression (12), revealing that viral expression patterns reflect the severity of disease. In addition, proliferation of the basal cells does not stop when the basement layer is complete in high-risk lesions, in contrast to low-risk lesions. The increased basal layer proliferation is correlated with beta-catenin activity and E6 levels (13, 14). What drives increased E6 activity in some infections is unknown: wound healing, episomal copy number, DNA methylation patterns, and host genetic background could all contribute and require further investigation. Nevertheless, these observations have the potential to tremendously improve existing cervical screening practice, which relies heavily on cytology. Though in vitro studies are invaluable for identifying candidate molecules involved in pathogenesis, conformation of their role often requires an in vivo approach. Dr Paul F. Lambert, University of Wisconsin Medical School, described transgenic mouse models of HPV where the human keratin promoter, hK14, was used to drive expression of HPV genes in stratified squamous epithelia (15). Previous studies had shown that expression of HPV genes was insufficient to cause cervical cancer in mice (15). Lambert's group hypothesized that estrogen was a cofactor for cervical cancer. They generated hK14-HPV16-estrogen receptor transgenic mice, which did develop cervical cancer (16-18). This may account for observations that >5 years of contraceptive pill usage and pregnancy increases the risk of women developing cervical cancer. HPV is not just associated with cancer of the cervix, however. Prof Iqbal Parker of the ICGEB, Cape Town, showed that papilloma viruses typically considered low-risk could be involved in fatal cancers of the oesophagus. Parker's group found that about 40% of oesophageal cancer (OC) patients had integrated HPV-11 and -39 DNA in their tumors (19). Brush biopsies of healthy people confirmed that HPV was enriched in OC patients. However, smoking, alcoholic home brew consumption and cooking on indoor fires in conjunction with certain Sulpho-transferase alleles were also risk factors associated with the development of OC in Africa (20). Whether HPV infection is an early or initiation event in OC development requires further investigation. The molecular alterations that direct progression from productive infection to HSILs in HPV infection are not fully understood. HPV oncoproteins alone are not capable of transformation, as shown in human keratinocytes in vitro (21) and murine cervical epithelia in vivo (15). Rather, papilloma virus-mediated oncogenesis requires supplementary genetic changes that occur over time following the initial infection. Whilst it is clear that integration of viral DNA into the host genome is crucial to HPV-induced tumor development (22, 23), whether this is a cause or consequence of wider-spread chromosome instability is not clear. Despite the activity of various oncogenic HPV proteins, viral DNA integration and cancer development does not often follow. The import of the host, including an ability to regulate viral gene expression in different tissues and to mount an effective immune response, is becoming increasingly apparent in determining the molecular basis of HPV-associated tumor progression. Dr Ethel Cesarman, Weill Cornell Medical College, described Human Herpes viruses and focused on KSHV, also known as Human Herpes Virus 8 (HHV8). KSHV causes a cancer often found in immunosuppressed individuals, such as those suffering from AIDS, including Karposi's sarcoma primary effusion lymphoma and multicentric Castleman's disease. There is >50% KSHV seroprevalence in sub-Saharan Africa but, as with HPV, the majority of individuals present with no disease. KSHV contains up to 90 ORF with at least 15 accessory genes. Among the accessory genes identified to date, ORF74, named KSHV G protein-coupled receptor (KSHV-GPCR), has become a major focus of investigation. Though it is expressed in a very small number of KS lesion spindle cells (24), it is known to be involved in cell transformation through induction of angiogenic cytokine expression (25). For example, vascular endothelial growth factor is one cytokine secreted from cells expressing KSHV-GPCR, which can induce proliferation and angiogenesis in KS tissue (26). Following the observation that proteins expressed by lymphomagenic viruses during latent infection activate NF-κB, Cesarman's group searched for a viral protein whose activity correlated with NF-κB. They identified vFLIP, an homologue of cFLIP proteins, and showed that anti-vFLIP siRNAs induced apoptosis in KSHV-positive cells only (27). Work is continuing to develop drugs that are vFLIP-specific, in the hope that these will prove effective anti-KSHV agents. During his lectures, Dr Ingemar Ernberg, of the Karolinska Institute in Stockholm, recoined the acronym EBV as "Every Bodies Virus" because >95% of humans are infected from childhood, though infections are typically subclinical. A member of the HHV family, EBV was initially isolated from a Burkitt's lymphoma cell line (28), though it was later found associated with a number of different tumors, such as undifferentiated nasopharyngeal carcinoma, Hodgkin's disease, nasal T cell lymphoma and gastric carcinoma. Ernberg described the four programs of latent viral gene expression (L0–L3) and how all 12 viral genes are expressed in L3, which is associated with cell proliferation (29). Therefore, Ernberg's group looked for the signal controlling the switch from L1 to L3. They showed that in L1, there was low expression of the viral gene EBNA1, but high levels of the cellular transcription factor Oct2, and vice versa in L3. Work is ongoing into deciphering the reason for a decrease in Oct2 levels that is concomitant with a switch to the L3 program of gene expression. Hopefully this will shed light on why EBV set points vary between individuals and, further, what differs between the B cells of those who appear immune to EBV infection and the majority of the population who are susceptible. Dr Aluisio Segurado, University of São Paulo, described HTLV-1, a retrovirus that can result in Adult T cell leukemia/lymphoma (ATLL) and HTLV-1-associated myelopathy (HAM/TSP). It is estimated that 15 to 20 million individuals are infected with HTLV-1 worldwide, yet the vast majority remain clinically asymptomatic--only 2–6% develop ATLL (30). The reason for the low frequency of HTLV-1-associated disease is unknown but Segurado hypothesises that host genetics and environmental factors play a role, rather than infection with different strains of HTLV. Tax (p40tax) is one of the HTLV-1 proteins that acts as a transcription activator in ATLL (31). However, the low incidence rate and the long latency period prior to development of ATLL suggests that, in addition to viral infection, accumulation of mutations in host genes is required for cellular transformation in vivo. Among the hepatitis viruses, HBV and HCV cause chronic infection leading to the development of cirrhosis and hepatocellular carcinoma (HCC). HBV is a DNA virus that integrates into the host genome. Dr Shahid Jameel, ICGEB, New Delhi, described how this integration deregulates expression of the viral protein HBx, which is able to induce HCC, either alone or in synergy with different cellular proteins (32). Jameel also discussed tumorigenesis induced by HCV: chronic immune-mediated inflammation and associated oxidative chromosomal DNA damage probably play a role. Interactions of viral proteins with pRb and p53 may also predispose to carcinogenesis (33). Although not all the viruses discussed induce the same pathway to cancer, some parallels can be drawn. A requirement for viral oncogenes indicates that the viruses play a crucial role in progression to malignancy. Both RNA and DNA tumor viruses promote growth of infected tissue by activation of cellular oncogenes and inactivation of tumor suppressor genes. Interestingly, many cellular oncogenes and tumor suppressor genes (e.g., p53, pRb) were identified through studies of RNA and DNA tumor viruses, respectively. After infection, these viruses can establish either latent (HPV, EBV, KSHV, HTLV-1) or chronic (HBV, HCV) infection. Activation from latent infection may result from changes in the cell environment, an accumulation of viral stress on the cell and/or a decreased ability of the host to maintain latency or clear infection. Such activation signals may be dependent on the host's genetic make-up (34) and epigenetic factors (35). Understanding why some individuals infected with tumor viruses do not develop cancer remains critical. It is anticipated that continuing research into what distinguishes 'normal' viral life cycles from the life cycles in malignant tissues, and the changes in host factors accompanying transformation, will address this problem. So far it has been demonstrated that changes in viral gene expression may accompany the development of high-risk lesions (e.g., HPV, EBV). But why are such changes occurring at a low frequency in infected populations? Are these changes in gene expression a result of a virus-host interaction disruption? Answering these questions requires a great deal more research, but certainly the speakers at the Summer School are on course to do just that. A detailed understanding of the molecular mechanisms governing virus infection and propagation is crucial to the development of antiviral strategies through identification of critical processes and drug targets. This was best exemplified at the Summer School by three speakers who are elucidating complimentary stages of the HIV life cycle: Ariberto Fassati, University College London, discussed nuclear import, Alessandro Marcello, ICGEB, Trieste, focused on integration and the spatial and temporal regulation of HIV-1 gene expression, and Hans-Georg Kräusslich, Universität Heidelberg, dealt with assembly and maturation. Although existing highly active antiretroviral therapy (HAART) can significantly reduce HIV-related illness, the emergence of drug resistant strains, toxic side effects and their ineffectiveness in latently infected cells has ensured that the development of novel HIV-1 therapies remains an important objective. Lentiviruses, such as HIV-1, have the ability to infect terminally differentiated nondividing cells and, therefore, require nuclear import. However, the HIV-1 reverse transcription complex (RTC) is larger than the nuclear pore diffusion size limit. In addition, the RTC is enriched for nucleic acids and so must overcome the hydrophobic exclusion of nuclear pore complexes and a concentration gradient of DNA to enter the nucleus. The manner in which HIV-1 resolves this dilemma is worth investigating as nuclear import is critical to HIV-1 transmission and AIDS pathogenesis. Dr A. Fassati is doing just that and his group has identified some novel mechanisms by which HIV-1 enters the nucleus. A number of viral elements have been shown to play a role in nuclear import e.g., the cPPT element, a nuclear localization signal within matrix and the viral proteins integrase (IN) and Vpr. Fassati's group was interested in identifying the host nuclear transport receptor responsible for HIV-1 import. They used purified RTC complexes in primary macrophages to show that importin 7 (imp7) is involved (36), an import receptor for RNA- and DNA-binding ribosomal and histone proteins, respectively. Further work revealed that the role of imp7 was HIV-specific, which correlated with its ability to bind HIV IN (37). RNAi-mediated knockdown of imp7 decreased import of DNA, but not RNA, indicating that reverse transcription is not a requirement for nuclear import (37). The DNA import-function of imp7 is likely hijacked by HIV-1 to facilitate import of the HIV-1 DNA genome, although it remains unclear whether the RTC remains intact throughout the nuclear import process. Fassati's group continued to search for other nuclear import pathways. They used nucleic acid dye-labeled RTCs and cells treated with digitonin (to permeabilise the plasma, but not the nuclear, membrane) to monitor nuclear import in the presence of various cytosolic extracts. The almost capable of inducing nuclear import of HIV-1 RTCs was found to contain showed that nuclear import of at least some of occurs in cells cellular pathway of unknown that HIV-1 to are present in HIV-1 and it is that the nuclear import of the RTC with host which have yet to be Fassati's work not only novel import for HIV-1, and thus, potential drug it has also our knowledge of cellular biology through identification of the tRNA nuclear import viral are early during HIV infection, which of the as are not susceptible to antiretroviral In addition, latent DNA can be to viral on of Therefore, understanding the molecular mechanisms governing latency and of viral expression is crucial to developing strategies at complete of the this Dr A. described his work into the of and chromosome on the of HIV-1 transcription and has shown that the histone the protein and histone is enriched at DNA can the on HIV-1 gene expression in from Whilst into the mechanisms underlying HIV-1 the remains as to how some cells to be latently with at the the propensity for HIV-1 to into active The is a highly and and it is now that active transcription is enriched at the nuclear group the latently infected cell cells developed by by and showed that along with viral transcription occurring at the nuclear the latent was at the conformation capture revealed an of the latent with a of chromosome 12 in which was on of transcription Although it be that latency is a result of a rare integration event into an gene at the nuclear work a in which integration into an active gene and of the T cell population is by a number of T cells becoming cells by a that is by the of the through epigenetic of this in an manner is an ongoing The host cell is a environment, so need to form in the but the to be into a new viruses have different to this In the is provided by the a with a of is During of the by in a of the to form a infectious virus with a of the of despite no in the size of the Dr provided an overview of his work the mechanisms of virus and the to studies revealed that following of the contains proteins assembled into a A was to contain more than the number present in the studies demonstrated that, in is in an in addition, contains The may to the by the as there is no evidence of as in the The of and its in the and stages may a of the prior to assembly of the The of is but such a be an drug the virus contains an how is the virus from the it was that assembled at the into that in with of the resulting by the cellular complex required for transport machinery group demonstrated that almost complete were only present at where was with their observations that the is of an a novel for the of HIV-1 is by assembly with early to drive The can therefore, considered a assembly occur than is microscopy, group was able to that an in at of HIV-1 early in assembly understanding of the role of host in HIV-1 to be an HIV-1 is by the HIV accessory protein However, and mouse HIV-1 through its to This has for the development of small models of HIV infection. The work of Kräusslich, Marcello, and Fassati that continued into the molecular mechanisms of HIV-1 replication development of effective an to all The immune system is important in the detrimental effects of HPV more lesions are in patients of HPV DNA in women is which may be due to changes in the immune Dr Doorbar described how the immune system to clear cells are not very effective as viral proteins are expressed at low levels in the lower epithelial In addition, HPV proteins E6 and E7 and respectively is also to play an role through of major Hence, without HPV-associated warts and can to It is also this in immune clearance of the virus that may predispose individuals to cancer. an HPV is Whilst the only the most predominant high-risk so that it is Nevertheless, of this reduce the cervical cancer of has the world over Prof the immune to this humans successfully following the of although the virus is to prior to The of a is of for a gene to a virus that is able to infect humans and cause to the protein are However, the virus is able to the immune system through and the the potential to cause of to virus infection in mice, but of did not This the that requires not which to be in also discussed the of to the antiviral immune described the in which with one and with not just but also to For example, with will not e.g., but will improve the primary to years This to the of B cells as and cells: T B cell not just B cell following primary to T cell a more response, including to This is important in as it the importance of not just the of but also the so as to was first reported in in and within a few the virus had to more than in South and Prof a detailed on his development of a His group has demonstrated that virus mice from fatal disease when with and mice expressing the viral with showed decreased inflammation and on The also induced T cell and Work is ongoing to the role of but gene expression studies regulation of the stress Whilst highly the is not yet considered for particularly the of and the risk of involves but this also as inactivation can be The of rather than in could improve the of without HIV has infected more than million people

The aim of this chapter is to study catastrophic pandemics which have occurred in the twentieth and twenty-first centuries and their disruptive impact on tourism mobility. A detailed study of past pandemics is conducted starting from the black death or bubonic plague of 1346 to the recent COVID-19 outbreak and effect of these diseases on the tourism and economy of the infected countries. Studies show that influenza pandemics will prove to be the most dangerous in future, and the next outbreak could occur from any of the 16 known HA (haemagglutinin) subtypes. Also, it is found that tourism itself has been responsible for spread of pandemic outbreaks as countries around the world put an enormous emphasis on increased growth of tourist numbers. Among recent pandemics, it was severe acute respiratory syndrome (SARS) that caused the major decrease in international tourist arrivals although for a short term. Such pandemics have a negative effect on tourism destinations by damaging their image and competitiveness, and as a result, leading to disruptions in mobility of tourists, with Asian countries being the most at risk of such disruptions. Therefore, the findings of this study stress the need for pre-crisis management to handle such outbreaks, better traveller tracking system to check infected persons and the need for tourism destinations to diversify their economies to reduce dependency on tourism.

The development of modern medicine has allowed us to conquer numerous infectious diseases; however, we human beings constantly face threats from novel infectious diseases that have been previously unrecognized. These so-called “emerging infectious diseases” are often caused by zoonotic pathogens, which mostly originate in wild animals (1, 2). Human diseases, such as AIDS, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), Ebola viral disease, and pandemic influenza are all caused by such pathogens. To cause zoonosis, the pathogens that originate in animals must cross the species barrier and transmit to humans. If these pathogens are able to efficiently transmit from human to human, a pandemic would result, endangering the lives of humans globally. Aquatic wild birds harbor a large gene pool of influenza A viruses that have been the source of influenza pandemics. Although influenza A viruses can infect a wide range of species, host restriction usually constrains their interspecies transmission; however, some mammalian-adaptive mutations have been identified in hemagglutinin (HA) and Polymerase Basic 2 (PB2) that allow avian influenza viruses to overcome the species barrier and become transmissible via the airborne route among ferrets (3). In addition, PB1 has been shown to confer to airborne transmission to H5N1 viruses (4). For over 25 y, Webster’s group has conducted surveillance of avian viruses at Delaware Bay, New Jersey, and has investigated the biological properties of the isolated H1N1 avian viruses in mammalian models (5, 6). Surprisingly, some of the H1N1 avian isolates transmitted via the airborne route in a ferret model without prior adaptation (5⇓–7), suggesting no adaptive mutations were required for these viruses to become transmissible. By comparing the genomes of the transmissible and nontransmissible viruses, Zanin et al. (7) identify differences in the PB2, PB1, PB1-F2, PA-X, NS1, and NEP genes … [↵][1]1To whom correspondence should be addressed. Email: yoshihiro.kawaoka{at}wisc.edu. [1]: #xref-corresp-1-1

Introduction: Viruses are spread from one individual to another. In respect to the mode of transmission, the majority of them enter the human body by the inward breath of infective respiratory beads. Upper Respiratory Tract Infections (URTI) are infections of the body’s respiratory tract, which include the sinuses, nose, throat, airways, and lungs. The influenza virus has four key structural antigens: the internal Ribonucleoprotein (RNP), the viral envelope Matrix (M), and two surface Glycoproteins (GP), Neuraminidase (NA) and Haemagglutinin (HA). A respiratory virus called swine flu/H1N1 evolved and spread widely around the world. H1N1 outbreaks with various virus strains were noted before the most recent severe pandemic, which occurred in 2009. Seasonal outbreaks that are extremely infrequent since the 2009 pandemic the influenza strain have occurred. Aim: To assess the prevalence and level of cytokines in H1N1- infected patients with signs and symptoms of respiratory distress. Materials and Methods: This is a cross-sectional study of viral molecular, immunological and epidemiological parameters. The study was done at the Indian Council of Medical Research (ICMR) at the Department of Health Research (DHR), Virus Research and Diagnostic Laboratory (VRDL), Department of Microbiology, Burdwan Medical College and Hospital (BMCH), Burdwan, India. The duration of the study was seven months, from January 2022- July 2022. Samples in Viral Transport Medium (VTM) were collected from suspected influenza patients with mild or severe Acute Respiratory Distress Symptoms (ARDS) and Influenza Like Symptom (ILS). The RNA samples were isolated and the nucleic acid purified from samples was screened by Real-time Polymerase Chain Reaction (RT-PCR). The extracted clinical Ribonucleic Acid (RNA) samples are then converted into Complementary Deoxyribonucleic acid (cDNA). The HA gene sequences of endemic swine influenza A virus (H1N1) and sequences from a panel of human and avian type A influenza virus strains, including the type A human seasonal strains, were retrieved from the GenBank database in the National Centre for Biotechnology Information (NCBI) portal. The Enzymelinked Immunoassay (ELISA) method was used to measure the proinflammatory cytokines Interleukin (IL)-8, IL2 and antiinflammatory cytokine IL10, as well as the IL3 concentration level in severe Acute Respiratory Distress Syndrome (ARDS) +Immediate Life Support (ILS) positive H1N1 infected patients. Analysis of all the data was performed by Statistical Package for Social Sciences (SPSS) software version 22. Results: In this study, all samples (n=120) were examined using RT-PCR, which revealed that 53 samples were infected with the Influenza A Virus (IAV). Among the total positive 24 were males and 29 were females. the average concentration of IL3 was 1749.49 pg/mL. The increase in IL8 was not as big as the increase in IL2 and IL3. IL8 was identified as a significant proinflammatory factor during angiogenesis, or uncontrolled cell growth. Conclusion: The prevalence of H1N1 infection was found to be high in children under the age of ten. The concentration of IL-3 in H1N1-infected patients’ samples was higher than the concentrations of the other three cytokines.

Induction of broadly reactive influenza antibodies increases susceptibility to autoimmunity.

Cetylpyridinium chloride (CPC) reduces zebrafish mortality from influenza infection: Super-resolution microscopy reveals CPC interference with multiple protein interactions with phosphatidylinositol 4,5-bisphosphate in immune function

Surface plasmon resonance (SPR) is used to measure hemagglutinin (HA) binding to domain-swapped Cyanovirin-N (CV-N) dimer and to monitor interactions between mannosylated peptides and CV-N's high-affinity binding site. Virus envelope spikes gp120, HA, and Ebola glycoprotein (GP) 1,2 have been reported to bind both high- and low-affinity binding sites on dimeric CVN2. Dimannosylated HA peptide is also bound at the two low-affinity binding sites to an engineered molecule of CVN2, which is bearing a high-affinity site for the respective ligand and mutated to replace a stabilizing disulfide bond in the carbohydrate-binding pocket, thus confirming multivalent binding. HA binding is shown to one high-affinity binding site of pseudo-antibody CVN2 at a dissociation constant (KD) of 275 nM that further neutralizes human immunodeficiency virus type 1 (HIV-1) through oligomerization. Correlating the number of disulfide bridges in domain-swapped CVN2, which are decreased from 4 to 2 by substituting cystines into polar residue pairs of glutamic acid and arginine, results in reduced binding affinity to HA. Among the strongest interactions, Ebola GP1,2 is bound by CVN2 with two high-affinity binding sites in the lower nanomolar range using the envelope glycan without a transmembrane domain. In the present study, binding of the multispecific monomeric CV-N to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein is measured at KD = 18.6 µM as compared with nanomolar KD to those other virus spikes, and via its receptor-binding domain in the mid-µ-molar range.

BackgroundNeutralizing antibodies (NAbs) are important for protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reinfection. In this study, two assays that are correlated with NAbs were compared: the haemagglutination test (HAT) and the surrogate virus neutralization test (sVNT). MethodsThe specificity of the HAT was compared with the sVNT, and the sensitivity and persistence of antibodies in patients with varying severity of illness was assessed in a cohort of 71 patients at 4–6 weeks and 13–16 weeks. The kinetics were assessed in the first, second, and third weeks in patients with varying severity of acute illness. ResultsThe specificity of the HAT was >99%, and sensitivity was similar to the sVNT. The levels of HAT were significantly and positively correlated with those of the sVNT (Spearman's r = 0.78, P < 0.0001). Patients with moderate and severe illness had higher HAT titres when compared to those with mild illness. Six of seven patients with severe illness had a titre of >1:640 during the second week of illness, whereas only five of 31 patients with a mild illness had a titre of >1:160 in the second week of illness. ConclusionsSince the HAT is a simple and very cheap assay to perform, it would be ideal to use as an indicator of NAbs in resource-poor settings.

Social distancing, mask‐wearing, and travel restrictions during the COVID‐19 pandemic have significantly impacted the spread of influenza viruses. The objectives of this study were to analyze the pattern of influenza virus circulation with respect to that of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) in Bulgaria during the 2021–2022 season and to perform a phylogenetic/molecular analysis of the hemagglutinin (HA) and neuraminidase (NA) sequences of representative influenza strains. Influenza infection was confirmed using real‐time reverse transcription polymerase chain reaction in 93 (4.2%) of the 2193 patients with acute respiratory illness tested wherein all detected viruses were subtyped as A(H3N2). SARS‐CoV‐2 was identified in 377 (24.3%) of the 1552 patients tested. Significant differences in the incidence of influenza viruses and SARS‐CoV‐2 were found between individual age groups, outpatients/inpatients, and in the seasonal distribution of cases. Two cases of coinfections were identified. In hospitalized patients, the Ct values of influenza viruses at admission were lower in adults aged ≥65 years (indicating higher viral load) than in children aged 0–14 years (p < 0.05). In SARS‐CoV‐2‐positive inpatients, this association was not statistically significant. HA genes of all A(H3N2) viruses analyzed belonged to subclade 3C.2a1b.2a. The sequenced viruses carried 11 substitutions in HA and 5 in NA, in comparison to the vaccine virus A/Cambodia/e0826360/2020, including several substitutions in the HA antigenic sites B and C. This study revealed extensive changes in the typical epidemiology of influenza infection, including a dramatic reduction in the number of cases, diminished genetic diversity of circulating viruses, changes in age, and seasonal distribution of cases.

Introduction. After two seasons of absence and low circulation, influenza activity increased significantly in the winter of 2022-2023. This study aims to characterize virological and epidemiological aspects of influenza infection in Bulgaria during the 2022-2023 season and perform a phylogenetic/molecular analysis of the hemagglutinin (HA) and neuraminidase (NA) sequences of representative influenza strains.Hypothesis/Gap Statement. Influenza A and B viruses generate new genetic groups/clades each season, replacing previously circulating variants. This results in increased antigenic distances from current vaccine strains. Strengthening existing influenza surveillance is essential to meet the challenges posed by the co-circulation of influenza and SARS-CoV-2.Methodology. We tested 2713 clinical samples from patients with acute respiratory illnesses using a multiplex real-time RT-PCR kit (FluSC2) to detect influenza A/B and Severe acute respiratory syndrome coronavirus-2(SARS-CoV-2) simultaneously. Representative Bulgarian influenza strains were sequenced at the WHO Collaborating Centres in London, UK, and Atlanta, USA.Results. Influenza virus was detected in 694 (25.6 %) patients. Of these, 364 (52.4 %), 213 (30.7 %) and 117 (16.9 %) were positive for influenza A(H1N1)pdm09, A(H3N2) and B/Victoria lineage virus, respectively. HA genes of the 47 influenza A(H1N1)pdm09 viruses fell into clades 5a.2. and 5a.2a.1 within the 6B.5A.1A.5a.2 group. Twenty-seven A(H3N2) viruses belonging to subclades 2b, 2a.1, 2a.1b and 2a.3a.1 within the 3C.2a1b.2a.2 group were analysed. All 23 sequenced B/Victoria lineage viruses were classified into the V1A.3a.2 group. We identified amino acid substitutions in HA and NA compared with the vaccine strains, including several substitutions in the HA antigenic sites.Conclusion. The study's findings showed genetic diversity among the influenza A viruses and, to a lesser extent, among B viruses, circulating in the first season after the lifting of anti-COVID-19 measures.