Avian flu: «for whom the bell tolls¼?

The family Orthomyxoviridae consists of 9 genera, including Alphainfluenza virus, which contains avian influenza viruses. In two subtypes H5 and H7 besides common low-virulent strains, a specific type of highly virulent avian virus have been described to cause more than 60% mortality among domestic birds. These variants of influenza virus are usually referred to as «avian influenza virus¼. The difference between high (HPAI) and low (LPAI) virulent influenza viruses is due to the structure of the arginine-containing proteolytic activation site in the hemagglutinin (HA) protein. The highly virulent avian influenza virus H5 was identified more than 100 years ago and during this time they cause outbreaks among wild and domestic birds on all continents and only a few local episodes of the disease in humans have been identified in XXI century. Currently, a sharp increase in the incidence of highly virulent virus of the H5N1 subtype (clade h2.3.4.4b) has been registered in birds on all continents, accompanied by the transmission of the virus to various species of mammals. The recorded global mortality rate among wild, domestic and agricultural birds from H5 subtype is approaching to the level of 1 billion cases. A dangerous epidemic factor is becoming more frequent outbreaks of avian influenza with high mortality among mammals, in particular seals and marine lions in North and South America, minks and fur-bearing animals in Spain and Finland, domestic and street cats in Poland. H5N1 avian influenza clade h2.3.4.4b strains isolated from mammals have genetic signatures of partial adaptation to the human body in the PB2, NP, HA, NA genes, which play a major role in regulating the aerosol transmission and the host range of the virus. The current situation poses a real threat of pre-adaptation of the virus in mammals as intermediate hosts, followed by the transition of the pre-adapted virus into the human population with catastrophic consequences.

Two Phylogenetic Cohorts of the Nucleocapsid Protein NP and Their Correlation with the Host Range of Influenza A Viruses.

Influenza A virus has a wide natural areal among birds, mammals, and humans. One of the main regulatory adaptors of the virus host range is the major NP protein of the viral nucleocapsid. Phylogenetic analysis of the NP protein of different viruses has revealed the existence of two phylogenetic cohorts in human influenza virus population. Cohort I includes classical human viruses that caused epidemics in 1957, 1968, 1977. Cohort II includes the H1N1/2009pdm virus, which had a mixed avian-swine origin but caused global human pandemic. Also, the highly virulent H5N1 avian influenza virus emerged in 2021 and caused outbreaks of lethal infections in mammals including humans, appeared to have the NP gene of the second phylogenetic cohort and, therefore, by the type of adaptation to human is similar to the H1N1/2009pdm virus and seems to possess a high epidemic potential for humans. The data obtained shed light on pathways and dynamics of adaptation of avian influenza viruses to humans and propose phylogenetic algorithm for systemic monitoring of dangerous virus strains to predict epidemic harbingers and take immediate preventive measures.

The Unique Genome of the Virus and Alternative Strategies for its Realization.

Dedicated to the 130th anniversary of Dmitry Ivanovsky's discovery of the virus kingdom as a new form of biological life. The genome of some RNA-containing viruses comprises ambipolar genes that are arranged in stacks (one above the other) encoding proteins in opposite directions. Ambipolar genes provide a new approach for developing viral diversity when virions possessing an identical genome may differ in its expression scheme (strategy) and have distinct types of progeny virions varying in the genomic RNA polarity and the composition of proteins expressed by positive- or negative-sense genes, the so-called ambipolar virions. So far, this pathway of viral genome expression remains hypothetical and hidden from us, like the dark side of the Moon, and deserves a detailed study.

[130th anniversary of virology].

130 years ago, in 1892, our great compatriot Dmitry Iosifovich Ivanovsky (18641920) discovered a new type of pathogen viruses. Viruses have existed since the birth of life on Earth and for more than three billion years, as the biosphere evolved, they are included in interpopulation interactions with representatives of all kingdoms of life: archaea, bacteria, protozoa, algae, fungi, plants, invertebrates, and vertebrates, including the Homo sapiens (Hominidae, Homininae). Discovery of D.I. Ivanovsky laid the foundation for a new science virology. The rapid development of virology in the 20th century was associated with the fight against emerging and reemerging infections, epidemics (epizootics) and pandemics (panzootics) of which posed a threat to national and global biosecurity (tick-borne and other encephalitis, hemorrhagic fevers, influenza, smallpox, poliomyelitis, HIV, parenteral hepatitis, coronaviral and other infections). Fundamental research on viruses created the basis for the development of effective methods of diagnostics, vaccine prophylaxis, and antiviral drugs. Russian virologists continue to occupy leading positions in some priority areas of modern virology in vaccinology, environmental studies oz zoonotic viruses, studies of viral evolution in various ecosystems, and several other areas. A meaningful combination of theoretical approaches to studying the evolution of viruses with innovative methods for studying their molecular genetic properties and the creation of new generations of vaccines and antiviral drugs on this basis will significantly reduce the consequences of future pandemics or panzootics. The review presents the main stages in the formation and development of virology as a science in Russia with an emphasis on the most significant achievements of soviet and Russian virologists in the fight against viral infectious diseases.

The cleavage of spike protein НА0→НА1/HA2 by trypsin permits activation of the M2 channel without its proteolytic cleavage in the influenza A virus.

M2 plays numerous regulatory roles in influenza A virus infection confirming the old adage: "a little body often harbors a great sense". The comment here demonstrates that a small viral protein M2, having 14 kD m.w. and situating in the virion at a minor amount of only about 40 molecules per virus particle is resistant to trypsin at concentrations initiating the HA0 cleavage and virus infectivity activation. A mechanism involving a programmed disassembly by cascade-type transmembrane signaling of the HA-M2-M1-RNP cooperation during virus entry into the infected cell is proposed.

Novel Negative Sense Genes in the RNA Genome of Coronaviruses.

The coronavirus family consists of lipid-containing envelope viruses that have a single-stranded RNA genome that encodes 25-30 proteins in different viruses by the mechanism of positive-polarity strategy. In addition, extended open reading trnslation frames (ORFs, genes) located in a negative-sense orientation were found in the genomes of coronaviruses. The size of negative-sense genes varies in the range of 150-450 nt, which corresponds to polypeptides encoded by negative-polarity genes (negative gene proteins, NGP) with m. m. 5-30 × 103 kDa. Coronaviruses show marked differences from virus to virus in the number of negative genes detected. These negative-sense genes in the coronavirus genome allow this family to be considered as viruses developing an ambisense genome strategy.

[Method for evaluating the neuraminidase activity of receptordestroying enzyme (RDE) compounds using the influenza virus.]

INTRODUCTION: The classic hemagglutination inhibition reaction (RTGA) is used to determine the level of antiviral antibodies in human and animal serum specimens. During the performance of RTGA the tested sera must be treated with a receptor-destroying enzyme (RDE) to remove serum glycans that degrade the accuracy of the RTGA results. To optimize the amounts of RDE compounds used, it is necessary to know their real neuraminidase activity. This article describes a simple and economical method for testing the neuraminidase activity of receptordestroying compounds using standard reagents and laboratory equipment. Aims of investigation. Design of an improved simple and convenient method for evaluating the neuramin1idase activity using the flu virus. MATERIAL AND METHODS: Here, we propose a convenient method for evaluating the activity of neuraminidase by double-fold dilution procedure with human or animal erythrocytes followed by hemagglutination assay with influenza A virus. RESULTS AND DISCUSSION: The method is based on the ability of neuraminidase to hydrolyze sialic acid residues on the cell surface of erythrocytes, that deprives red blood cells to be agglutinated with the flu virus, since these sialic glycans provide virus attachment and hemagglutination. CONCLUSION: The designed method allows the accurate measurement of the receptor-destroying (neuraminidase) activity of RDE compounds and the comparison of the compounds with each other. This test is necessary to optimize the RTGA protocol when monitoring blood sera of animals and humans after influenza infection and/or Acute Respiratory diseases (ARD). The designed method can be included in the guidelines of regulations for the RTGA protocol, which is used in different laboratories to monitor the epidemic process of influenza and ARD infections.

Molecular Targets in the Chemotherapy of Coronavirus Infection.

In the pathogenesis of the infectious process in the respiratory tract by SARS, MERS, and COVID-19 coronaviruses, two stages can be distinguished: early (etiotropic) and late (pathogenetic) ones. In the first stage, when the virus multiplication and accumulation are prevalent under insufficient host immune response, the use of chemotherapeutic agents blocking the reproduction of the virus is reasonable to suppress the development of the disease. This article considers six major chemotherapeutic classes aimed at certain viral targets: inhibitors of viral RNA polymerase, inhibitors of viral protease Mpro, inhibitors of proteolytic activation of viral protein S allowing virus entry into the target cell, inhibitors of virus uncoating in cellular endosomes, compounds of exogenous interferons, and compounds of natural and recombinant virus-neutralizing antibodies. In the second stage, when the multiplication of the virus decreases and threatening pathological processes of excessive inflammation, acute respiratory distress syndrome, pulmonary edema, hypoxia, and secondary bacterial pneumonia and sepsis events develop, a pathogenetic therapeutic approach including extracorporeal blood oxygenation, detoxification, and anti-inflammatory and anti-bacterial therapy seems to be the most effective way for the patient's recovery.

Unique Bipolar Gene Architecture in the RNA Genome of Influenza A Virus.

The genome of influenza A virus consists of eight single-stranded negative-polarity RNA segments. The eighth segment (NS) encodes the anti-interferon protein NS1 (27 kDa) and the nuclear export protein NEP (14 kDa) via the classic negative-sense strategy. It also contains an additional positive-sense open reading frame that can be directly translated into the negative strand protein 8 (NSP8; 18-25 kDa in different strains). The existence of three or more genes of the opposite polarity in the same locus of a single-stranded RNA appears to be a unique ("economical") type of gene architecture in living organisms. In silico analysis of genomes of human and animal influenza A viruses revealed that the NSP8 gene had emerged in the influenza A virus population about 100 years ago ("young" gene) and is highly evolutionary variable. The obtained experimental data suggest that NSP8 gene is expressed in the infected animals, which strengthens the concept of bipolar (ambisense) strategy of the influenza A virus genome. The high variability of the NSP8 protein suggests that the "young" NSP8 gene is in the process of functional optimization. Further accumulation of mutations may alter the functions of mature NSP8 protein and lead to the emergence of mature bipolar influenza A virus with unexpected properties that would be threatening for humans and animals.

NSP Protein Encoded in Negative NS RNA Strand of Influenza A Virus Induces Cellular Immune Response in Infected Animals.

Infection of mice with influenza A viruses led to the formation of clones of lymphocytes that specifically recognizes viral domains in the central zone of the NSP protein (amino acid positions 83-119). Computer analysis of the primary structure of the NSP protein showed the presence of T-cell epitopes in the central part of the NSP molecule. The findings indicate that the viral NSP gene is expressed in the infected animals and verify the concept of the bipolar strategy (ambisense strategy) of the influenza A virus genome.

Biochemical Variations in Cytolytic Activity of Ortho- and Paramyxoviruses in Human Lung Tumor Cell Culture.

Human lung cancer cells (Calu-3 line) were studied for the development of apoptosis, necrosis, and autophagy in response to infection with ortho- and paramyxoviruses. Biochemical pathways underlying various mechanisms of cell death differed for different viruses. When infected with murine Sendai paramyxovirus, Calu-3 cells demonstrated typical necrotic features such as cell swelling (but not shrinkage), lack of chromatin DNA laddering, of caspase 3 and 8 activation, and of apoptotic cleavage of poly(ADP-ribose) polymerase (PARP) protein; an activation of antiapoptotic protein kinase Akt was also revealed. In contrast, infection with avian influenza virus A/FPV/Rostock/34 (H7N1 subtype) or Newcastle disease virus (NDV, avian paramyxovirus) caused the development of typical apoptotic markers such as cell shrinkage, ladder-type chromosomal DNA fragmentation, caspase 3 and 8 activation, and proteolytic cleavage of PARP in the absence of Akt activation. Notably, no upregulation of p53 protein phosphorylation was observed in all infected cells, which indicates that p53 is not involved in the virus-induced death of Calu-3 cells. Cell death caused by the influenza virus was accompanied by overstimulation of autophagy, whereas no stimulation of autophagy was observed in the NDV-infected cells. Infection with Sendai virus caused moderate stimulation of autophagy, which suggests that the mechanism of the virus-induced cell death and the balance between autophagy and cell death in infected cancer cells depend on the virus type and might significantly differ even for closely related viruses. Therefore, an optimal strategy for oncolytic virus-mediated destruction of tumor cells in cancer patients requires selection of the most appropriate oncolytic virus based on the mechanism of its cytolytic action in a particular type of tumor.

Negative-sense virion RNA of segment 8 (NS) of influenza a virus is able to translate in vitro a new viral protein.

It was shown that full-length virion RNA of segment 8 of influenza virus A/Aichi/2/68 (H3N2) can initiate the synthesis of two major polypeptides with molecular weights of 23 and 13 kD and a minor polypeptide with a molecular weight of 19 kDa, which specifically reacted with the antibodies to the 30-membered peptide of the central part of the NSP protein of influenza A virus. Thus, the genomic-polarity RNA of segment 8 of influenza virus A has a translational template function. These data provide further confirmation of the concept of the bipolar (ambisens) strategy of functioning of the influenza A virus genome.

Paramyxoviruses activation by host proteases in cultures of normal and cancer cells.

Multiplication of paramyxovirus Sendai and Newcastle disease virus (NDV) was studied in cultures of normal and tumor cells. Production of noninfectious virus with uncleaved F0 was observed in canine kidney cell line MDCK (line H) and its derivatives carrying tetracycline-regulated expression of transmembrane protease HAT or TMPRSS2 with trypsin-like cleavage specificity. Under tetracycline induction, a cleavage F0 (65 kD)>F1 (50 kD)+F2(15 kD) and production of infectious virus were observed in these cell cultures. Under tetracycline induction, the additional subunit 38K (m.w. 38 kDa) of the F protein was detected both in infected MDCK-HAT cells and in newly synthesized Sendai virus in addition to F0, F1 and F2, indicating thereby a second HAT-sensitive proteolytic site in the F0 molecule. Highly infectious virus containing cleaved F1+F2 was produced in cultures of cancer cells Caco-2 and H1299. Virus Sendai synthesized in H1299 cells contained 38 K subunit indicating a cleavage of the F0 at a second site by H1299 host cell proteases. Levels of cleaved F1+F2 and infectious virions were higher at the late stage of infection in cancer cells, suggesting thus the induction of virus-activating proteases in Caco-2 and H1299 cells under infection with paramyxoviruses. NDV virus was found to induce more rapid death of cancer cells Caco-2 than Sendai virus. Cooperatively, the obtained data show that cancer cells in distinction to nonmalignant cells can synthesize protease(s) activating infectivity of paramyxoviruses. Thus, they are more vulnerable to paramyxovirus infection than normal cells.

Intravirion cohesion of matrix protein M1 with ribonucleocapsid is a prerequisite of influenza virus infectivity.

Influenza virus has two major structural modules, an external lipid envelope and an internal ribonucleocapsid containing the genomic RNA in the form of the ribonucleoprotein (RNP) complex, both of which are interlinked by the matrix protein M1. Here we studied M1-RNP cohesion within virus exposed to acidic pH in vitro. The effect of acidification was dependent on the cleavage of the surface glycoprotein HA. Acidic pH caused a loss of intravirion RNP-M1 cohesion and activated RNP polymerase activity in virus with cleaved HA (HA1/2) but not in the uncleaved (HA0) virus. The in vitro acidified HA1/2 virus rapidly lost infectivity whereas the HA0 one retained infectivity, following activation by trypsin, suggesting that premature activation and release of the RNP is detrimental to viral infectivity. Rimantadine, an inhibitor of the M2 ion channel, was found to protect the HA1/2 virus interior against acidic disintegration, confirming that M2-dependent proton translocation is essential for the intravirion RNP release and suggesting that the M2 ion channel is only active in virions with cleaved HA. Acidic treatment of both HA0 and HA1/2 influenza viruses induces formation of spikeless bleb-like protrusion of ~ 25 nm in diameter on the surface of the virion, though only the HA1/2 virus was permeable to protons and permitted RNP release. It is likely that this bleb corresponds to the M2-enriched and M1-depleted focus arising from pinching off of the virus during the completion of budding. Cooperatively, the data suggest that the influenza virus has an asymmetric structure where the M1-mediated organization of the RNP inside the virion is a prerequisite for infectious entry into target cell.

Asymmetric structure of the influenza A virus and novel function of the matrix protein M1.

Influenza virus is an enveloped virus. It comprises two major modules: external lipoprotein envelope and internal ribonucleoprotein (RNP) containing the genomic negative-strand RNA. Lipoprotein envelope contains four vital proteins: hemagglutinin (HA), neuraminidase (NA), transmembrane ionic channel M2, and minor amounts of nuclear export protein NEP. RNP contains RNA and four polypeptides: major nucleocapsid protein NP and three polymerase subunits PB1, PB2, PA. Both modules are linked with each other by matrix M1 maintaining the virus integrity. According to the structural function, NP and M1 are predominant in virus particle in the amounts of 1000 and 3000 molecules, respectively. In addition to the structural function, M1 plays a role in regulation of intracellular and nuclear migration of viral RNP and virus assembly, referred as budding process, at the plasma membrane in infected cells. The bipolar structure of the influenza virus characterized by asymmetric location of RNP and nonregular distribution of M1 and M2 inside the virion is reviewed. The role of M1 in maintaining the asymmetric structure of the virus particle and regulation of RNP transport inside virus particle is considered. First experimental data confirming (i) intravirion RNP transport and its outside exit directed by the M1 and (ii) the importance of this process in virus uncoating and initiation of infection in target cell are discussed. A novel class of antiviral agents activating ATP-ase of the early endosome compartment in the target cell is discussed.

Abnormal Morphological Vesicles in Influenza A Virus Exposed to Acid pH.

Vesicles on the virion surface, which continued the lipoprotein membrane but had no spikes of virus glycoproteins hemagglutinin (HA) and neuraminidase (NA), were detected. These vesicles and virus particles were 18±7 and 103±12 nm in diameter, respectively, and, as a rule, one vesicle was found per virion. The locus of the external protrusion in the virion presumably corresponded to the site of virus budding during assembly in infected cell free from HA and NA spikes outside and M1 matrix protein inside, but enriched with ionic channel protein M2. Particles with vesicles constituted ~3-10% of the virus population produced in MDCK-H culture and containing uncleaved HA0 hemagglutinin. The content of vesicular virions increased slightly after trypsin cleavage HA0→HA1+HA2 and reached 10-15%. Exposure of the virus in acid medium (pH 4.3) led to a drastic increase of vesicular virions - to 60-80% for HA0 and HA1+HA2 virus. This was paralleled by changes in contrast permeability (for phosphotungstic acid). HA0 virions remained contrast-impermeable, while HA1+HA2 particles let the contrast in through the vesicles detected on the surface and were rapidly destroyed after incubation in acid medium. Hence, cleavage of the surface glycoprotein HA0 into HA1 and HA2 stimulated the acid-dependent permeability of the lipid membrane and led to attenuation of the ribonucleoprotein and protein matrix M1 contacts inside the virion.

[pH-dependent rearrangements in the influenza A virus].

The Influenza virus possesses two modules: internal ribonucleoprotein (RNP) containing the viral genome RNA and external lipid envelope with transmembrane ionic channel protein M2 and embedded glycoproteins hemagglutinin (HA) and neuraminidase (NA) forming surface spike ends. These modules are combined in a whole virion by the matrix protein M1. The effect of the acidic pH 4,2-4,5 on the influenza virus grown in MDCK-H cells was tested. The A/Aichi/68 (H3N2) virus synthesized in MDCK-H cells was shown to contain uncleaved HA0 (m.w. 78 kD) and provide low infectivity. This virus was resistant to acidic medium and non-permeable to the phosphotungsten acid (PTA) used in electron microscopy as a contrast stain, and did not reduce infectious potential after acidic treatment. The trypsin-activated virus containing cleaved HA1 (56 kD)+HA2 (22 kD) was sensitive to acidic exposition resulting in the appearance of permeability to PTA, reduction of infectivity, enhancement of the M1-RNP interlink. These data indicate that the structural form of the cleaved HA1 +HA2 surface hemagglutinin coordinates a transmembrane interaction between surface and internal virus components.

[Pathogenic effect of pandemic influenza virus H1N1 under replication in cultures of human cells].

The propagation of the pandemic influenza virus H1N1 in cultures of bronchial (Calu-3) and intestinal (Caco-2) differentiated epithelial cells of human origin was studied. The canine epithelial cell lines, MDCK-H and MDCK-2, were comparatively tested. The two human cell lines were found to be highly sensitive to the influenza pandemic strains A/Hamburg/05/09 and A/Moscow/501/2011 and maintained their replication without addition of trypsin to culture medium. Virus strains of seasonal influenza H1N1, such as A/Moscow/450/2003, A/Memphis/14/96, and laboratory strain A/PR/8/34, multiplied in these human cells in similar manner. The intracellular cleavage HA0-->HA1+HA2 by the host virus-activating protease (IAP) occurred in both human cell lines under infection with each influenza virus H1N1 including pandemic ones. Comparatively, this cleavage of all influenza H1N1 virus strains appeared to be either undetectable or low-detectible in MDCK-H and MDCK-2, respectively, thereby implying low levels of active IAP in these cells. Multiplication of pandemic and seasonal influenza H1N1 viruses in Calu-3 and Caco-2 cells caused cytopathic effect, which was accompanied with low autophagy and apoptosis events. These data allow recommending human cell lines, Calu-3 and Caco-2, for optimized isolation and passaging of clinical strains of Influenza pandemic viruses H1N1.

Influenza A virus proteins NS1 and hemagglutinin along with M2 are involved in stimulation of autophagy in infected cells.

The NS1 protein of influenza A virus is known to downregulate apoptosis early in infection in order to support virus replication (O. P. Zhirnov, T. E. Konakova, T. Wolff, and H. D. Klenk, J. Virol. 76:1617-1625, 2002). In the present study, we analyzed the development of autophagy, another mechanism to protect cells from degradation that depends on NS1 expression. To this end, we compared autophagy in cells infected with wild-type (WT) influenza virus and virus lacking the NS1 gene (delNS1 virus). The results show that in WT-infected cells but not in delNS1 virus-infected cells, synthesis of the autophagy marker LC3-II, the lipidated form of microtubule light chain-associated protein LC3, is stimulated and that LC3-II accumulates in a perinuclear zone enriched with double-layered membrane vesicles characteristic of autophagosomes. Transfection experiments revealed that NS1 expressed alone was unable to upregulate autophagy, whereas hemagglutinin (HA) and M2 were. Proteolytic cleavage of HA increased autophagy. Taken together, these observations indicate that NS1 stimulates autophagy indirectly by upregulating the synthesis of HA and M2. Thus, it appears that NS1, besides downregulating apoptosis, is involved in upregulation of autophagy and that it supports the survival of infected cells by both mechanisms.

[Evolutionary changes in the NA and HA genes of H3N2 influenza virus in the Moscow Region during 2003-2009].

During the winter 2009 outbreak in the Moscow Region, H3N2 influenza viruses were isolated from the nasopharyngeal washes of patients via their propagation in the human intestinal (Caco-2) and bronchial (Calu-3) epithelial cell cultures maintaining the proteolytic cleavage of HA0--> HA1+HA2 and multicycle virus replication. Analysis of the nucleotide sequences of virus RNA indicated that the 2009 viruses differed from those isolated in 2003 in 14 and 21 amino acids of the neuraminidase (NA) and hemagglutinin (HA) genes, respectively. The NA gene was 1762 nucleotides long whereas the 2003 isolates had a deletion of 66 nucleotides (22 amino acids) in the stalk region (short-stalk NA genotype) of viruses. The NA gene of the 2009 and 2003 isolates possessed an amino acid profile characterized for oseltamivir- and zanamivir-susceptible viral strains. The HA gene of the 2009 viruses contained an N-glycosylation site at Asn181 (an analog to Asn 65 numbering from a signal peptide), which correlated with the long-stalk NA gene. The 2009 viruses had Phe209 in the HA receptor binding center whereas the 2003 isolates possessed Ser209, which correlated with their differences in HA activity. Phylogenetic analysis showed that the NA genes of the 2003 and 2009 Moscow strains were located in the same genetic clade with a single common precursor while their HA genes were diverged in more genetic distance and located in different clades. Viral distribution in the phylogenetic tree indicated that the Moscow strains isolated in 2009 were not direct ancestors of those isolated in 2003; and during the period of 2003 to 2009, H3N2 influenza virus with a short-stalk NA genotype was substituted for a migrant virus possessing a long-stalk NA gene.