Murine respirovirus

The virus is responsible for a highly transmissible respiratory tract infection in mice, hamsters, guinea pigs, rats,[7] and occasionally marmosets[8] with infection passing through both air and direct contact routes. The virus can be detected in mouse colonies worldwide,[9] generally in suckling to young adult mice. A study in France reported antibodies to SeV in 17% of mouse colonies examined.[10] Epizootic infections of mice are usually associated with a high mortality rate, while enzootic disease patterns suggest that the virus is latent and can be cleared over the course of a year.[1] Sublethal exposure to SeV can promote long-lasting immunity to further lethal doses of SeV.[11] There are no scientific studies using modern detection methods, which would identify SeV as an infectious agent that can cause disease in humans or domestic animals.

Structure of Sendai virus virion

Inbred and outbred mouse and rat strains have very different susceptibility to Sendai virus infection: The 129/J mice tested were approximately 25,000-fold more sensitive than SJL/J mice.[12] C57BL/6 mice are highly resistant to the virus, while DBA/2J mice are sensitive.[13] C57BL/6 mice showed slight loss of body weight after SeV administration, which returned to normal later. Only 10% mortality rate was observed in C57BL/6 mice after the administration of very high virulent dose of 1*10⁵ TCID50.[14] It was shown that resistance to the lethal effects of Sendai virus in mice is genetically controlled and expressed through control of viral replication within the first 72 hours of infection.[13] Treatment of both strains with exogenous IFN before and during viral infection led to an increase in survival time in C57BL/6 mice, but all animals of both strains ultimately succumb to SeV caused disease.[15] If a mouse survives a SeV infection, it develops a lifelong immunity to subsequent viral infections.[16]

There are SeV-resistant F344 rats and susceptible BN rats.[17]

The virus is a powerful immunomodulator; it can cause a strong immune response. However, SeV can act in both directions: it can activate or suppress the immune response depending on the type of cell and host.

SeV is able to suppress the IFN production and the IFN response pathways as well as inflammation pathway.

Sendai virus P gene encodes a nested set of proteins (C', C, Y1 and Y2), which are named to collectively as the C proteins (see the section "genome structure" below). C proteins of SeV are able to suppress apoptosis.[18] The antiapoptotic activity of the C proteins supports SeV infection in the host cells.

SeV can suppress cell defense mechanisms by inhibiting the interferon production. The virus prevents the stimulation of type 1 IFN production and subsequent cell apoptosis in response to virus infection by inhibiting the activation of IRF-3.[19][20][21] Two virus proteins: C and V are mainly involved in this process.

Inhibition of stimulation of type 1 IFN production also occurs by suppressing the phosphorylation of IRF7 by viral C protein. The C protein interacts with IKKα serine / threonine kinase and prevents the phosphorylation of IRF7. Thus, C-protein inhibits a pathway that includes a Toll-like receptor (TLR7) and TLR9-induction of IFN-alpha, which is specific for plasmacytoid dendritic cells.[22]

V protein from SeV and many other paramyxoviruses has been shown to directly bind MDA5 and inhibit its activation of the IFN promoter.[23][24] V protein can also bind RIG-I and TRIM25. This binding prevents downstream RIG-I signaling to the mitochondrial antiviral signaling protein (MAVS) by disrupting TRIM25 -mediated ubiquitination of RIG-I.[25]

Human Parainfluenza Virus type 1 (HPIV1), which is a close relative of SeV and (in contrast to SeV) a successful human pathogen, does not express V proteins, only C proteins. So, all needed functions provided by V in SeV can be provided by C in HPIV1. Thus, C and V have these "overlapping functions" because of the multi-faceted nature of host defense that can be countered at so many places, and exactly how well and where will in part explain host restriction.[26]

SeV can attenuate cell defense mechanisms and allow itself to escape from host innate immunity by inhibiting the interferon response pathway in addition to inhibiting the interferon production. C protein of the virus suppresses the signal transduction pathways of interferon alpha/beta (IFN-α/β) and IFN-γ by binding to the N-terminal domain of STAT1.[27][28]

C protein of the virus inhibits the production of nitric oxide (NO) by murine macrophages[29][30] that has cytotoxic activity against viruses.[31]

V protein of SeV also suppresses the production of interleukin-1β, by inhibiting the assembly of the inflammasome NLRP3.[32]

• Sneezing
• Hunched posture
• Respiratory distress
• Porphyrin discharge from eyes and/or nose
• Lethargy
• Failure to thrive in surviving babies and young rats
• Anorexia

SeV induces lesions within the respiratory tract, usually associated with bacterial inflammation of the trachea and lung (tracheitis and bronchopneumonia, respectively). However, the lesions are limited, and aren't indicative solely of SeV infection. Detection, therefore, makes use of SeV-specific antigens in several serological methods, including ELISA, immunofluorescence, and hemagglutination assays, with particular emphasis on use of the ELISA for its high sensitivity (unlike the hemagglutination assay) and its fairly early detection (unlike the immunofluorescence assay).[33]

In a natural setting, the respiratory infection of Sendai virus in mice is acute. From the extrapolation of the infection of laboratory mice, the presence of the virus may first be detected in the lungs 48 to 72 hours following exposure. As the virus replicates in the respiratory tract of an infected mouse, the concentration of the virus grows most quickly during the third day of infection. After that, the growth of the virus is slower but consistent. Typically, the peak concentration of the virus is on the sixth or seventh day, and rapid decline follows that by the ninth day. A fairly vigorous immune response mounted against the virus is the cause of this decline. The longest period of detected presence of the virus in a mouse lung is fourteen days after infection.[34]

Eaton et al. advises that, when controlling an outbreak of SeV, disinfecting the laboratory environment and vaccinating the breeders, as well as eliminating infected animals and screening incoming animals, should clear the problem very quickly. Imported animals should be vaccinated with SeV and placed in quarantine, while, in the laboratory environment, breeding programs should be discontinued, and the non-breeding adults isolated for two months.[35]

Currently, there is no scientific data obtained using modern detection methods that would identify SeV as an infectious - disease causing agent for humans. Modern methods for the identification of pathogenic microorganisms have never detected SeV in pigs or other domestic animals, despite the isolation of other paramyxoviruses.[36][37][38][39][40][41] Consequently, it is recognized that Sendai virus disease causing infection is host restrictive for rodents and the virus does not cause disease in humans[42] or domestic animals, which are natural hosts for their own parainfluenza viruses.

In 1952, Kuroya and his colleagues attempted to identify an infectious agent in human tissue samples at Tohoku University Hospital, Sendai, Japan. The samples were taken from the lung of a newborn child that was affected by fatal pneumonia. The primary isolate from the samples was passaged in mice and subsequently in embryonated eggs.[43][44] The isolated infectious agent was later called Sendai virus, which was used interchangeably with the name “Hemagglutinating Virus of Japan”. Kuroya and his colleagues were convinced that they isolated the virus, which is a new etiological agent for human respiratory infections. Later in 1954, Fukumi and his colleagues at the Japan National Institute of Health put forward an alternative explanation for the origin of the virus. It was suggested that the mice used to passage the virus were infected with the mouse virus. Thus, mouse virus was later transferred to embryonated eggs, isolated and finally named the Sendai virus.[45] This explanation of Fukumi, pointing to the mouse rather than the human origin of the virus, has been supported by numerous scientific data later. The historical aspects of the Sendai virus isolation and controversy behind it are well described in the review.[1] Thus, for some time, it was erroneously assumed that Sendai virus is human disease causing pathogen.[46] The incorrect assumption that the virus was isolated from human infectious material is still reported by Encyclopedia Britannica[47] and by ATCC in the description of the history of the viral isolate Sendai/52. It was also believed that the virus could cause disease not only in humans but also in pigs, because antibodies to the virus were often found in their organisms during the swine epidemic in Japan in 1953-1956. High incidence of seropositivity to the virus was observed in pigs in 15 districts of Japan.[46] An explanation was later found for this widespread detection of antibodies (see the section below). Yet, despite overwhelming evidence that indicate that SeV is host restrictive rodent pathogen, in some veterinary manuals and safety leaflets, SeV is still listed as a virus that can cause disease in pigs. Similar information is provided by Encyclopedia Britannica.[47] In reality, the multiple isolates of paramyxoviruses in pigs, using modern nucleic acid sequencing methods, have never been identified as SeV.[36][37][38][39][40][41]

All viruses in the Paramyxoviridae family are antigenically stable, therefore porcine parainfluenza 1,[38][39] which is a close relative of SeV, most likely, has cross-reactive antibodies with SeV. This cross-reactivity may explain why SeV antibodies were found in pigs, and why it was thought that SeV was the etiological causative agent of pigs disease in Japan in 1953-1956.[46] Human parainfluenza virus type 1, also shares common antigenic determinants with SeV and triggers the generation of cross-reactive neutralizing antibodies.[48] This fact can explain wide spread detection of SeV antibodies in humans in the 1950-1960s.[46] After experimental SeV infection the virus can replicate and shed from the upper and lower respiratory tract of African green monkeys and chimpanzees, but it is not causing any disease.[49]

SeV can overcome antiviral mechanisms in some of its natural hosts (rodents), but the virus is ineffective in overcoming these mechanisms in some virus resistant rodents and in other organisms that are not natural hosts of the virus such as humans.[50] Normal cells respond to viral infections by activating specific defense mechanisms. Virus-induced cell death due to apoptosis, which limits the spread of infection, is one such mechanism. In addition to stimulating the production of IFN, SeV triggers the production of other cytokines that provide protection against viruses (see "Stimulation of the secretion of interferons (IFN) type I and II" and "Stimulation of the secretion of immunostimulating antitumor chemokines and cytokines" sections below).

SeV can activate the ubiquitously expressed interferon regulatory factor 3 (IRF-3), by triggering its post-translational phosphorylation in human cells. IRF-3, is activated by phosphorylation on a specific serine residue, Ser³⁹⁶.[51] There is also some evidence that demonstrates that SeV activates not only IRF-3, but the transcription factors NF-κB[52] and also IRF-7.[53] This stimulates IFN production and IFN signal transduction pathway that finally should lead to cell apoptosis.[54]

SeV replication triggers activation of MAPK/ERK pathway (also known as the Ras-Raf-MEK-ERK pathway) in a RIG-I-dependent manner in dendritic cells (DC) and in fibroblasts. RIG-I-mediated activation of this pathway by SeV results in type I IFN production.[55]

RIG-1

IRF7-mediated induction of IFN-α by SeV requires both RIG-I and mitochondrial antiviral-signaling protein (MAVS) expression.[56] MAVS is also needed for SeV induction of IκB kinase (IKK), IRF3 and IFN-β in human cells.[57]

MDA5

Melanoma differentiation-associated antigen 5 (MDA5) has been shown to be an important participant in the antiviral SeV response and IFN type I production.[58]

Monocytes[59] and dendritic cells[60] produce IFN alpha/beta in response to SeV stimulation. However, plasmacytoid dendritic cells (pDC) ,dispite inability to be infected by SeV,[61] produce higher level of IFN-1 compared to monocytes and monocyte-derived dendritic cells in response to SeV. This happens most likely due to the higher levels of constitutively expressed IRF-7 in pDC compared to monocytes and monocyte-derived dendritic cells.[62]

The recognition of SeV by pDC happens through TLR7 activation and requires transport of cytosolic viral replication products into the lysosome by the process of autophagy. Moreover, for pDC, autophagy was found to be required for these cells production of IFN-α.[61]

Among conventional DCs only two subsets: CD4⁺ and CD8α−CD4− “double negative”[63] dendritic cells produce IFN-α and IFN-β in response to SeV infection. However, all conventional DC subsets including CD8α⁺ can be infected with SeV.[64] SeV can replicate to high titers in human monocyte-derived DCs.[65] However, pDCs are not producing a significant number of SeV virions after infection.[61] UV-irradiated SeV triggers lower levels of IFN-α production in pDC compared to alive virus.[61]

Human mast cell infection SeV induces the expression of type 1 IFN.[66]

SeV can stimulate and/or inhibit the IFN-beta response pathway depending on the type of cell and host. Multiple examples of IFN-beta protecting cells from SeV infection are described It has been shown that SeV infects human lung fibroblasts MRC-5 and induces the release of IFN-beta into the culture medium from infected cells. Pretreatment of these cells with IFN-beta inhibits the replication of SeV.[50]

A similar IFN-beta protection against the virus has been observed for some human malignant cells that maintain the IFN response pathway. HeLa cells can be infected with SeV, however, incubation of these cells with IFN-beta causes inhibition of SeV replication.[67] Multiple interferon stimulated genes (ISG) were identified as being required for this inhibition including IRF-9, ZNFX1, TRIM69, NPIP, TDRD7, PNPT1 and so on.[67] One of this genes TDRD7 was investigated in more detail. The functional TDRD7 protein inhibits the replication of SeV and other paramyxoviruses, suppressing autophagy, which is necessary for productive infection with these viruses.[67]

IFN also triggers the expression of IFN induced Ifit2 protein that is involved in protecting mice from SeV through an as yet unknown mechanism.[68]

Using human embryonic kidney cells (HEK 293T) it has been shown that SeV can stimulate production of a pattern recognition receptor NLRC5, which is a cytosolic protein expressed mainly in hematopoetic cells.[69] This production activates the cryopyrin (NALP3) inflammasome.[70] Using human monocytic cell line-1 (THP-1) it has been shown that SeV can activate signal transduction by mitochondrial antiviral-signaling protein signaling (MAVS), which is a mitochondria-associated adaptor molecule that is required for optimal NALP3-inflammasome activity. Through MAVS signaling SeV stimulates the oligomerization of NALP3 and triggers NALP3-dependent activation of caspase-1[71] that, in turn, stimulates caspase 1-dependent production of inteleukine -1 beta (IL-1β).[72]

SeV is a very effective stimulant of expression of human beta-defensin-1 (hBD-1). This protein is a member of the beta-defensin family of proteins that bridges innate and adaptive immune responses to a pathogen infection.[73] In response to SeV infection, the production of hBD-1 mRNA and protein increases 2 hours after exposure to the virus in purified plasmacytoid dendritic cells or in PBMC.[74]

Human mast cell infection with SeV induces an antiviral response with activation of expression of type 1 IFN, melanoma differentiation–associated gene 5 (MDA-5), retinoic acid-inducible gene-1 (RIG-1), and toll-like receptor 3 (TLR-3). In addition, SeV infection of mast cells induces the anti-viral proteins MxA and IFIT3.[66]

One of the great advantages of the Sendai virus as a potential oncolytic agent is its safety. Even though the virus is widespread in rodent colonies[1] and has been used in laboratory research for decades,[75] it has never been observed that it can cause human disease. Moreover, Sendai virus has been used in clinical trials involving both adults[48] and children[76] to immunize against human parainfluenza virus type 1, since the two viruses share common antigenic determinants and trigger the generation of cross-reactive neutralizing antibodies.The Sendai virus administration in the form of nasal drops in doses ranging from 5 × 10⁵ 50% egg infectious dose (EID50) to 5 × 10⁷ EID50 induced the production of neutralizing antibodies to the human virus without any measurable side effects.The results of these trials represent additional evidence of Sendai virus safety for humans.

For cancer studies, it is desirable that the oncolytic virus be non-pathogenic for experimental animals, but the Sendai virus can cause rodent disease, which is a problem for research strategies. Two approaches have been used to overcome this problem and make Sendai virus non-pathogenic for mice and rats. One of these approaches included the creation of a set of genetically modified attenuated viral strains. Representatives of this set were tested on model animals carrying a wide range of transplantable human tumors. It has been shown that they can cause suppression or even eradication of fibrosarcoma,[77][78] neuroblastoma,[79] hepatocellular carcinoma,[80] melanoma, squamous cell and prostate carcinomas.[81] Complete eradication of established gliosarcomas in immunocompetent rats has also been observed.[82] SeV constructs have also been created with a modified protease cleavage site in the F-protein. The modification allowed the recombinant virus to specifically infect cancer cells that expressed the corresponding proteases.[80]

Another approach of making Sendai virus non-pathogenic included the short-term treatment of the virions with ultraviolet light. Such treatment causes a loss of the virus replication ability. However, even this replication-deficient virus can induce the cancer cells death and stimulate anti-tumor immunity. It can trigger extensive apoptosis of human glioblastoma cells in culture, and it can efficiently suppress the growth of these cells in model animals.[83] The ultraviolet light treated virus can also kill human prostate cancer cells in culture[84] by triggering their apoptosis and eradicate tumors that originated from these cells in immunodeficient model animals.[85] Moreover, it can stimulate immunomodulated tumor regression of colon[86] and kidney cancers[87][88] in immunocompetent mice. Similar regressions caused by the replication-deficient Sendai virus have been observed in animals with transplanted melanoma tumors.[89][90]

Some cancer studies with non-rodent animals have been performed with the unmodified Sendai virus. Thus, after intratumoral injections of the virus, complete or partial remission of mast cell tumors (mastocytomas) was observed in dogs affected by this disease.[4] Short-term remission after an intravenous injection of SeV was described in a patient with acute leukemia treated in the Clinical Research Center of University Hospitals of Cleveland (USA) by multiple viruses in 1964.[91] It is also reported[3][92] that the Moscow strain of SeV[93] was tested by Dr. V. Senin[94] and his team as an anticancer agent in a few dozen patients affected by various malignancies with metastatic growth in Russia in the 1990s.[95] The virus was injected intradermally or intratumorally and it caused fever in less than half of the treated patients, which usually disappeared within 24 hours. Occasionally, the virus administration caused inflammation of the primary tumor and metastases. Clinical outcomes were variable. A small proportion of treated patients experienced pronounced long-term remission with the disappearance of primary tumors and metastases. Sometimes the remission lasted 5–10 years or more after virotherapy. Brief descriptions of the medical records of the patients that experiences long-term remission are presented in the patent.[95]

Some cell entry receptors for SeV (see the “Cell receptors” section below) are overexpressed in cancer cells, and their expression is important for carcinogenesis and/or metastasis. For example, the presence of Sialyl-Lewis^x antigen on the outer cell membrane correlates with invasion of malignant cells, tumor recurrence, and overall patient survival for an extremely wide range of cancers.[96] Expression of the Vim2 antigen is very important for the extravascular infiltration process of acute myeloid leukemia cells.[97] GD1a,[98] ganglioside is found in large quantities on the surfaces of breast cancer stem cells.[99] High cell surface expression of another ganglioside sialosylparagloboside /SPG/ NeuAcα2-3PG.[100] characterizes lymphoid leukemia cells.[101][102] GT1b, ganglioside is highly expressed on the outer membranes of brain metastases cells that originate from an extremely broad range of cancers.[103]

The fusion protein (F) of SeV is synthesized as an inactive precursor and is activated by proteolytic cleavage of the host cell serine proteases (see the section “Proteolytic cleavage by cellular proteases” below). Some of these proteases are overexpressed in malignant neoplasms. For example, transmembrane serine protease 2 (TMPRSS2), which is an F-protein-processing enzyme, is often overexpressed in prostate cancer cells. It is also overexpressed in some cell lines originating from various malignant neoplasms. Thus, it is highly expressed in bladder carcinoma RT4 urinary bladder carcinoma RT4, human colon carcinoma CaCo2[104] and breast carcinomas SK-BR-3, MCF7 and T-47d.[105] Another F-protein-protease is tryptase beta 2 (TPSB2). This protease (with alias such as tryptase-Clara and mast cell tryptase) is expressed in normal club cells, mast cells and in some cancers.[106] It’s especially high expression is observed in the human mast cell line HMC-1,[107][108] and in the human erythroleukemia cell line HEL.[109][107] Plasminogen (PLG), from which originates the mini-plasmin that can cleave the F-protein, is highly expressed in liver cancers.[110] Its expression is also increased in a wide range of other malignant neoplasms.[110] Factor X (F10) is frequently expressed in normal liver and in liver cancers.[111] SeV constructs were created with a modified protease cleavage site. The modification allowed the recombinant virus to specifically infect cancer cells that expressed the corresponding proteases, which can cleave a modified protease cleavage site.[112][77][80]

The interferon production and / or response system often malfunctions in malignant cells; therefore, they are much more vulnerable to infection with oncolytic viruses compared to normal cells.[113] Thus, cells belonging to three human cell lines, originated from variable malignancies, such as U937, Namalwa, and A549, retain their ability to become infected with SeV even after treatment with type 1 IFN. Broken interferon response system cannot protect these cells from SeV infection.[114]

The Namalwa cells, although there was some defect in the system of response to interferon, has been shown that SeV virus stimulates an expression of many genes involved in immune defense pathways, such as type I and type II IFN signaling, as well as cytokine signaling. Among the ten most virus-induced mRNAs are IFNα8, IFNα13, IFNβ, IFNλ:(L28α, IL28β, IL29), OASL, CXCL10, CXCL11 and HERC5.[115]

The host organism fights viral infection using various strategies. One such strategy is the production of neutralizing antibodies. In response to this production, viruses have developed their own strategies for spreading the infection and avoiding the inactivation by the host produced neutralizing antibodies. Some viruses, and in particular paramyxoviruses, can produce new virus particles by fusing infected and healthy host cells. This fusion leads to the formation of a large multi-nuclear structure (syncytium). Sendai virus, as a representative of Paramyxoviridae, uses this strategy to spread its infection (see the section “Directed cell fusion” below). The virus can fuse up to 50-100 cells adjacent to one primary infected cell. This multi-nuclear formation, derived from several dozens of cells, survives for several days and subsequently releases functional viral particles.

It has been demonstrated that the ability of a virus to destroy tumor cells increases along with an increase in the ability of the virus to form large multi-nuclear structures. The transfer of genes that are responsible for the formation of syncytium from the representative of Paramyxoviridae to the representatives of Rhabdoviridae or Herpesviridae makes the recipient viruses more oncolytic.[116][117] Moreover, the oncolytic potential of paramyxovirus can be enhanced by mutations in the fusion (F) gene protease-cleavage site, which allows the F-protein to be more efficiently processed by cellular proteases.[118] The introduction of the F gene of SeV in the form of a plasmid into the tumor tissue in mice by electroporation showed that the expression of the F gene increases the T cell infiltration of the tumor with CD4 + and CD8 + cells and inhibits tumor growth.[119] It was also shown in other similar experiments that cancer cells themselves, transfected with plasmids that encode viral membrane glycoproteins with fusion function, cause the collective death of neighboring cells forming syncytium with them. Recruitment of bystander cells into the syncytium leads to significant regression of the tumor.[120][121][122]

The virus triggers indirect immunomodulated death of malignant cells using a number of mechanisms, which are described in a published review.[3] The viral enzyme neuraminidase (NA), which has sialidase activity, can make cancer cells more visible to the immune system by removing sialic acid residues from the surface of malignant cells.[3] SeV activates natural killer cells (NK), cytotoxic T lymphocytes (CTL) and dendritic cells (DC). The secretion of interleukin-6, that is triggered by the virus, also inhibits regulatory T cells.

Type 1 and type II interferons have anticancer activity (see the "Function" section in the "Interferon" article). Sendai-virus-induced human peripheral blood leukocytes produce the interferon alpha (IFN-α)[123] and the interferon gamma (IFN-γ).[124][125] Interferon-beta (IFN-β) production in human fibroblast cells also occurs in response to SeV treatment.[126] Because of powerful IFN stimulating properties, before recombinant IFN became available for medical use, SeV was selected, among other viruses, for the industrial large-scale IFN production. A procedure involving inactivated SeV treatment of human peripheral blood leukocytes from donors’ blood was used for this production.[127] The SeV induced IFN-α consists from at least nine different sub types of IFN-α: 1a, 2b, 4b, 7a, 8b, 10a, 14c, 17b and 21b. Among these sub types IFN-α1 represents about 30% of total IFN-α.[128]

The Namalwa cells, which originated from human Burkitt lymphoma, has been shown that SeV virus stimulates an expression of IFNα8, IFNα13, IFNβ and IFN type III (IFN-lambda, IFNλ):(L28α, IL28β, IL29).[115]

UV-inactivated SeV can induce the production of type I IFN by some mouse dendritic cells,[129] and by some tumor cell lines.[85] However, UV-inactivated SeV triggers lower levels of IFN-α production in pDC compared to alive virus.[61] Most likely the glycoprotein HN is responsible for the effect of triggering type I IFN. It was shown that the HN of Paramyxoviruses is a potent inducer of type 1 IFN in human blood mononuclear cells[130] and the HN of SeV can induce type 1 IFN production in mouse spleen.[131]

It has been demonstrated that SeV can induce the production of IFN type III (IFN-lambda)[132] by human plasmacytoid dendritic cells.[133]

Sendai virus can induce the production of many cytokines that enhance cellular immune responses against cancer cells. SeV can stimulate the production of macrophage inflammatory protein-1α (MIB-1α) and –β (MIB-1β), RANTES (CCL5), tumor necrosis factor-alpha (TNF-alpha), tumor necrosis factor-beta (TNF-beta), interleukin-6 (IL-6 ), interleukin-8 (IL-8), interleukin-1 alpha (IL1A), interleukin-1 beta (IL1B), platelet-derived growth factor (PDGF-AB) and small concentrations of interleukin-2 (IL2) and GM-CSF.[125][134][135] Even plasmids that deliver the F-coding gene of SeV to tumor cells in model animals trigger the production of RANTES (CCL5) in tumor-infiltrated T-lymphocytes.[136]

SeV induces the production of B cell-activating factor by monocytes and by some other cells.[137]

Heat-inactivated SeV virus induces the production of IL-10 and IL-6 cytokines by dendritic cells (DC).[138] Most likely, F protein is responsible for this induction because reconstituted liposomes containing F protein can stimulate IL-6 production by DC. The production of IL-6 in response to SeV infection is restricted to conventional dendritic cells (DCs) subsets, such as CD4⁺ and double negative (dnDC).[64]

The UV-inactivated SeV (and likely the alive virus as well) can stimulate dendritic cells to secrete chemokines and cytokines such as interleukin-6, interferon-beta, chemokine (C-C motif) ligand 5, and chemokine (C-X-C motif) ligand 10. These molecules activate both CD8⁺ T cells as well as natural killer cells and attract them to the tumor. It has been shown that in cancer cell lines, UV-inactivated SeV triggers the production of an intercellular adhesion molecule -1 (ICAM-1, CD54), which is a glycoprotein that serves as a ligand for macrophage-1 antigen (Mac-1) and lymphocyte function-associated antigen 1 (LFA-1 (integrin)). Mac-1 and LFA-1 are receptors found on leukocytes. This induced production happens through the activation of nuclear factor-κB downstream of the mitochondrial antiviral signaling pathway and the retinoic acid-inducible gene I. The increased concentration of ICAM-1 on the surface of cancer cells, which is triggered by SeV, increases the vulnerability of these cells to natural killer cells.[139]

Elevated levels of cell membrane sialylation are associated with increased cancer cell potential for invasion and metastasis and with progression of the malignancy.[140][141][142][143][144][145] Some sialylation inhibitors can make cancer cells less malignant.[146][147][148]

One possible explanation for the relationship between increased sialylation and a malignant phenotype is that sialylation results in a thick layer of coating on the cell membrane that masks cancer antigens and protects malignant cells from immune surveillance. The activity and cytotoxicity of NK cells is inhibited by the expression of sialic acids on the tumor cell surface.[149] Removal of sialic acid residues from the surface of tumor cells makes them available to NK cells and cytotoxic T lymphocytes and, therefore, reduces their growth potential. Moreover, treating tumor cells with sialidase improves activation of NK cell secretion of IFN-γ.[149]

Some paramyxoviruses, including SeV encode and synthesize neuraminidase (sialidase), which can remove sialic acid residues from the surface of malignant cells. Hemagglutinin-neuraminidase (HN) is a single protein that induces hemagglutination and possesses neuraminidase (sialidase) activity. Neuraminidase (NA), a subunit of the HN protein, binds to and cleaves sialic acid from the cell surface.[150] NA also promotes cell fusion, which helps the nascent virions to avoid contact with host antibodies and thus enables the virus to spread within tissues.

Sialidase treatment of cells causes loss of sialic acid residues. This loss significantly increases the ability of malignant cells to activate cytotoxic T lymphocytes.[151] Variable sialidases can cause this effect,[151] including NA from Newcastle disease virus that have been shown to cleave 2,3-, 2,6-,[152] and 2,8-linkages between sialic acid residues.[153] In vitro, there was no significant difference between NAs from Newcastle disease virus, SeV and mumps virus[154] with respect to substrate specificity. These results suggest that treating a tumor with the virus results in desialylation of malignant cells, which contributes to increased anti-tumor immune surveillance. Therefore, the ability of SeV sialidase (NA) to remove sialic acid from the surface of malignant cells most likely helps to ensure the availability of tumor antigens for recognition by cytotoxic T lymphocytes.

Experiments with UV-inactivated SeV showed that NK cells are important in virus-mediated inhibition of tumor growth. This was shown in a mouse model of renal cancer, in which the anti-tumor effect of SeV was suppressed by reducing the number of NK cells by co-injection of specific antibodies.[155]

The activation of NK requires several receptors, among which are natural killer proteins 46 (NKp46) and 44 (NKp44). Studies have shown that the only paramyxovirus protein that activates NK is HN.[156] HN protein binding to NKp46 and/or NKp44 results in the lysis of cells whose surfaces display the HN protein or its fragments.[157][158] It can be assumed that NK activation and tumor suppression by UV-treated SeV[155] are caused by interaction between HN belonging to SeV, and NKp46 and/or NKp44 receptors belonging to NK cells.

SeV even after UV inactivation can cause tumor infiltration by dendritic cells (DCs) and CD4+ and CD8+ T and it also can cause enhancing of anti-tumor activity of these cells.[86] Most likely, viral hemagglutinin-neuraminidase protein, highly contributes to the effect (see "Neuraminidase (NA) removal of sialic acid from the surface of malignant cells stimulates natural killers cells and cytotoxic T lymphocytes" section above).This hypothesis is based on two observations. First, the functional hemagglutinin-neuraminidase protein of the oncolytic Newcastle disease virus (NDV), which is a relative of SeV, has been shown to enhance the tumor-specific cytotoxic response of CD8+ T-cells and to increase the activity of CD4+ T-helper cells.[158] Second, UV-inactivated NDV, which is unable to replicate, promotes anti-tumor CTL response as well as does intact NDV which is able to replicate.[158] Since the hemagglutinin-neuraminidase proteins of the SeV and NDV viruses are highly homologous, it is likely that the HN protein of the SeV virus can activate both CTL and natural killers cell responses. Most likely neuraminidase removal of sialic acid from the surface of malignant cells contributes to this effects.

UV-inactivated SeV can cause dendritic cells (DCs) to maturate and to infiltrate a tumor,[86] and ex vivo infection of DCs with recombinant SeV induces maturation and activation of DCs within 60 minutes.[159] When activated DCs that carry variants of recombinant SeV are administered, survival of animals injected with malignant melanoma,[160][161] colorectal cancer,[162] squamous cell carcinoma,[163] hepatic cancer, neuroblastoma, and prostate cancer[164] is significantly improved. It has been shown that the administration of such DCs prior to tumor cell injection prevents metastasis of neuroblastoma and prostate adenocarcinoma to the lungs.[165][166]

SeV can replicate to high titers in human monocyte-derived DCs.[65] With the multiplicity of infection of 2, approximately 1/3 of the DCs begin to express encoded SeV proteins 8 hours after infection. This proportion increases to 2/3, 24 hours and decreases to 1/3, 48 hours after infection. SeV demonstrates high cytopathic effect on DCs; the virus can kill a third of DC even with a very low multiplicity of infection such as 0.5. Most important observation is that SeV infection triggers DC maturation, which is manifested in DC cell surface markers composition. The virus increases the expression of class I and class II molecules of the major histocompatibility complex (MHC) (HLA-A, HLA-B, HLA-C and HLADR), CD83, as well as costimulatory molecules CD40 and CD86.[167]

Experiments with animal models have shown that, even after UV inactivation, SeV can block T-cell-mediated regulatory immunosuppression in tumors. The blocking mechanism is associated with the stimulation of SeV inactivated virions of interleukin 6 (IL-6) secretion by mature DCs. These effects lead to the eradication of most model tumors and inhibit the growth of the rest.[86] It has been shown that F protein alone can trigger IL-6 production in DC in a fusion-independent manner.[136]

Recombinant SeV variants has been constructed by introducing new genes and/or by deleting some viral genes such as F, M, and HN from the SeV genome,[170][171][172] SeV constructs have also been created with a modified protease cleavage site.[112][77][80]

A set of different recombinant SeV constructs was created for non-invasive imaging of the virus infection in animals. The constructs allow to study dynamics of SeV spread and clearance. One construct, rSeV-GFP4, which is commercially available, can deliver a green fluorescent protein (GFP4) to a cell. Another one rSeV-luc(M-F*) – luciferase.[173][174]

One of the latest applications of SeV-based vectors is the reprogramming of somatic cells into induced pluripotent stem cells.[175][176][177][112] The resulting iPSCs became trans-gene free.[178] The system for such reprogramming is commercially available from ThermoFisher Scientific as CTS™ CytoTune™-iPS 2.1 Sendai Reprogramming Kit, Catalog number: A34546.[179]

Virion structure is well described in a published review.[1] Sendai virus is an enveloped virus: its outer layer is a lipid envelope, which contains glycoprotein hemagglutinin-neurominidase (HN)[180] with two enzymatic activities (hemagglutinating and neuraminidase).[181] Hemagglutinin (H) serves as a cell attachment factor and membrane fusion protein. Neuraminidase (NA) is a sialidase that cleaves and removes sialic acid from the surface of a host cell. This cleavage promotes the fusion of the viral lipid envelope with the cell outer membrane.

In the lipid envelope of the virus located a fusion protein (F),[182] which is also a glycoprotein that ensures the virus entry into a host cell after viral adsorption. Under the lipid membrane is a matrix protein (M);[183] it forms the inner layer of the virus envelope and stabilizes it structure. The SeV virion also contains the nucleocapsid core, which is composed of the genomic RNA, the nucleocapsid protein (NP),[184] the phosphoproteins (P)[185] and the large protein (L)[186] that is the catalytic subunit of RNA-dependent RNA polymerase (RDRP). C-protein, which is translated from an alternative reading frame of the P-coding mRNA, is also associated with a viral capsid.[187]

The SeV F protein is a type I membrane glycoprotein that is synthesised as an inactive precursor (F₀) that must be activated by proteolytic cleavage at residue arginine-116.[188] After the cleavage F₀ precursor yields two disulfide-linked subunits F₁ and F₂.[189] Paramyxoviruses use different host cell proteases to activate their F-proteins. Sendai virus uses activating proteases, which are serine endopeptidases represented by tryptase beta 2-(TPSB2), which has aliases such as tryptase II, tryptase Clara, club cells tryptase, mast cells tryptase,[190][191][192][193] trypsin 1 (PRSS1),[194] mini-plasmin (PLG)[195] and transmembrane serine protease 2 (TMPRSS2).[196] Most likely, blood clotting factor X (F10) is capable to cleave and activate SeV F₀.[197][198][199] It is possible that other, not yet identified cellular proteases, can also process the F₀ protein of SeV.

Sendai virus genome structure The positions of translation initiation sites for products of the alternative reading frame of the P-coding mRNA are indicated.

The SeV genome is non-segmented, negative-sense RNA, of about 15.384 n. in length, and contains the noncoding 3’ leader and 5’ trailer regions, which are about 50 nucleotides in length.[188][200] As in other respiroviruses from Paramyxoviridae family, in SeV they work as cis-acting elements essential for replication. A 3’ leader sequence acts as a transcriptional promoter. Between these non-coding regions are located six genes, which encode the nucleocapsid (NP) protein, phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase (HN) and large (L) protein in this order from the 3’ terminus.[188][200] Intergenomic regions between these genes are three nucleotides long as in other respiroviruses. Additional proteins, which are frequently called non structural or accessory proteins can be produced from the P gene, using alternative reading frames.[188][201] The Sendai virus P/C mRNA contains five ribosomal initiation sites between positions 81 and 201 from the 5' end. One of these sites initiates in the P open reading frame, whereas four others initiate a nested set of C proteins (C', C, Y1, Y2).[202][201][203] These C proteins are initiated in the + 1 reading frame to that of P at different translation starting sites. Sendai virus uses ribosome shunting to express Y1 and Y2 proteins that initiate at the fourth and fifth start sites on the P/C mRNA (respectively).[203] Three additional SeV proteins are also encoded by P/C mRNA. Two of these proteins V and W are products of RNA editing, at codon 317 of the mRNA - G residues are added co-transcriptionally, (+one G residue for V and +two G for W).[204] The third - X protein is represented by 95 amino acids of the C terminal of the P protein and independently initiated by ribosomes.[205] All these non-structural proteins have several functions, including the organization of viral RNA synthesis and helping the virus to infect rodent cells by escaping host innate immunity (see "The mechanism of viral immunosuppression in natural hosts" section above).[204] It has also been found that C protein facilitates budding of virus-like particles[206] and small amounts of C protein are associated with a viral capsid.[187]

The strains of respiroviruses, avulaviruses, and the majority of rubulaviruses, that have HN in their envelopes, use sialic acids as their cell entry receptors. SeV, as a representative of respiriroviruses, uses lectin and the molecules containing sialic acids residues.

Three SeV receptors are represented by molecules that are clusters of differentiation.[207]

The structures of some of these receptors are available for visualization through SugarBindDB - a resource of glycan-mediated host–pathogen interactions.[224] Others are available through KEGG Glycan Database,[225] PubChem compound database,[226] and TOXNET database (toxicology data network) of US National Library of Medicine.[227]

Since SeV receptors are potential biomarkers for evaluation of the vulnerability of malignant cells to the virus, it is important to measure their expression level. Cellular expression of glycoproteins can be evaluated by various methods, which include RNA and protein measurements. However, cellular expression of gangliosides, which are sialic acid-containing glycosphingolipids, can not be evaluated by these methods. Instead it can be measured using anti-glycan antibodies, and despite the large collection of such antibodies in a community resource database, they are not always available for each ganglioside.[228] Therefore, indirect measurement of ganglioside expression by quantifying the levels of fucosyltransferases and glycosyltransferases that complete glycan synthesis is an alternative.There is evidence that expression of these enzymes and the production of gangliosides strongly correlate.[102]

At least four representatives of fucosyltransferases and several glycosyltransferases including sialyltransferases are responsible for the synthesis of gangliosides that can serve as SeV receptors. All of these proteins are often overexpressed in various tumors, and their expression levels correlate with the metastatic status of the tumor and the shorter life span of the patients. Thus, these enzymes are also potential biomarkers of SeV-oncolytic infectivity

One recognized feature of the Sendai virus, shared with members of its genus, is the ability to induce syncytia formation in vivo and in vitro in eukaryotic cell cultures.[237] The formation of syncytium helps the virus to avoid neutralizing antibodies of the host organism during the spread of infection.The mechanism for this process is fairly well understood and is very similar to the fusion process employed by the virion to facilitate cellular entry. The activities of the receptor binding hemagglutinin-neuraminidase protein is solely responsible for inducing close interaction between the virus envelope and the cellular membrane.

However, it is the F protein (one of many membrane fusion proteins) that, when triggered by local dehydration[238] and a conformational change in the bound HN protein,[239] actively inserts into the cellular membrane, which causes the envelope and the membrane to merge, followed shortly by virion entry. When the HN and F protein are manufactured by the cell and expressed on the surface, the same process may occur between adjacent cells, causing extensive membrane fusion and resulting in the formation of a syncytium.[240]

This behavior of SeV was utilized by Köhler and Milstein, who published an article in 1975 outlining a revolutionary method of manufacturing monoclonal antibodies. In need of a reliable method to produce large quantities of a specific antibody, the two merged a monoclonal B cell, exposed to a chosen antigen, and a myeloma tumor cell to produce hybridomas, capable of being grown indefinitely and of producing significant amounts of an antibody specifically targeting the chosen antigen. Though more efficient methods of creating such hybrids have since been found, Köhler and Milstein first used Sendai virus to create their revolutionary cells.[5]

All Sendai virus strains belong to the same serotype. The origin of many strains of SeV was described in 1978.[46] Some additional strains such as Ohita[254] and Hamamatsu[255] were described later. Ohita and Hamanatsu strains were isolated from separate epidemics in laboratory mice.[256][257] According to the personal memory of Alisa G. Bukrinskaya, who has co-authored numerous publications related to SeV along with Prof. Viktor M. Zhdanov, starting in 1961,[258] the Moscow strain of SeV[93] was obtained by Prof. Viktor M. Zhdanov of the Ivanovsky Institute of Virology from Japan in the late 1950s or early 1960s,[258] It is reported[259] that the BB1 strain[260] derived from the Moscow virus strain.[93] The strain BB1 was given to the researches of Institute of Viral Disease Control and Prevention, Beijing, China by researches of Ivanovsky Institute of Virology, Moscow, Russia in 1960s.[259]

A field SeV isolate, that is attenuated through egg-passages, is less virulent for mouse respiratory cells.[261] Therefore, the strains that were isolated from animals a few decades ago and went through multiple passages in eggs are less virulent for mice in comparison with the strains that are fresh field isolates.

Defective interfering (DI) genomes or defective viral genomes (DVGs) are replication defective viral RNA products generated during viral infections by many types of viruses, including SeV.[262][263][264] A single amino acid substitution in a nucleoprotein (NP) causes an increased production rate of DI genomes in the SeV Cantell strain, which is known for its particularly strong induction of interferon beta (IFN-β) during viral infection.[265] It has been shown that DI are responsible for this strong IFN-β induction.[266]

*The sequence of Enders strain is available from the US patent