Viruses Supplementary Material PDF

**Viruses** Parts written in small characters belong to facultative requirements! IV. **CLASSIFICATION - VIRUS FAMILIES** **SLIDE 1 Baltimore classes** **SLIDES 2-11 HERPESVIRUSES**: There are 3 subfamilies of herpesviruses: \(1) α-Herpesviridae: Herpes simplex virus type 1 and 2 (HSV1-2), vari...

**Viruses** Parts written in small characters belong to facultative requirements! IV. **CLASSIFICATION - VIRUS FAMILIES** **SLIDE 1 Baltimore classes** **SLIDES 2-11 HERPESVIRUSES**: There are 3 subfamilies of herpesviruses: \(1) α-Herpesviridae: Herpes simplex virus type 1 and 2 (HSV1-2), varicella-zoster virus (VZV) \(2) β-Herpesviridae: Human cytomegalovirus (HCMV) \(3) γ-Herpesviridae: Epstein-Barr virus (EBV) **HSV-1**: facial, labial and ocular lesions; **HSV-2**: genital lesions; **VZV**: chickenpox and shingles; **HCMV**: infectious mononucleosis; **EBV**: cofactor in human cancers More than 100 herpesviruses have been isolated from a range of hosts that includes mammals, birds, fish, reptiles, amphibians and mollusks. Eight of these viruses are human viruses. A notable characteristic of herpesviruses is that, once they have infected a host, they often remain as persistent infections for the lifetime of the host. These infections are often latent infections, which can be reactivated from time to time, especially if the host becomes immunocompromised. Both primary and reactivated herpesvirus infections can either be asymptomatic or can result in disease of varying severity. The outcome depends on the interplay between the particular virus and its host, and especially on the immune status of the host. There are eight herpesviruses known in man, and most adults in the world are persistently infected with most of them. ***Herpes simplex viruses 1 and 2*** (HSV-1 and HSV-2) initially infect epithelial cells of the oral or genital mucosa, the skin or the cornea. The virus may enter neurons and may be transported to their nuclei, where they may establish latent infections. **HSV-1** commonly infects via the lips or the nose between the ages of 6 and 18 months. A latent infection may be reactivated if, for example, the host becomes stressed or immunosuppressed. Reactivation results in the production of virions which are transported within the neuron to the initial site of infection, where they cause productive infection in epithelial cells, resulting in a cold sore. Occasionally there may be serious complications such as encephalitis, especially in immunocompromised hosts. **HSV-2** is the usual causative agent of genital herpes, which is a sexually transmitted disease. In newborn babies infection can result in serious disease. ***Varicella-zoster virus*** Infection with varicella-zoster virus usually occurs in childhood and causes varicella (chickenpox), when the virus spreads through the blood to the skin, causing a rash. It may also spread to nerve cells, where it may establish a latent infection. The nerves most often affected are those in the face or the trunk, and these are the areas most commonly affected in zoster (shingles) when a latent infection is reactivated. ***Human cytomegalovirus*** In the vast majority of infections with human cytomegalovirus symptoms are either absent or they are mild. In a pregnant woman, however, the virus can infect the placenta and then the foetus, for which the consequences may be serious. In other individuals damage develops at a later stage; the damage may be manifest in a number of ways, such as hearing loss and mental retardation. Human cytomegalovirus can also cause severe disease (e.g. pneumonitis, hepatitis) in immunocompromised patients such as those with AIDS, those who have received treatment for cancer and those who are immunosuppressed because they have received an organ transplant. ***Epstein-Barr virus*** Epstein-Barr virus (EBV) is transmitted in saliva. Epithelial cells are infected first then the infection spreads to B cells, which are the main host cell type for this virus. More than 90 per cent of people become infected with EBV, usually during the first years of life, when infection results in few or no symptoms. In developed countries some individuals do not become infected until adolescence or adulthood. A proportion of these individuals develop infectious mononucleosis (glandular fever), commonly called 'the kissing disease' by doctors. EBV is associated with a number of tumors in man. **The herpesvirus virion** Herpesviruses have relatively complex virions composed of a large number of protein species organized into three distinct structures: capsid, tegument and envelope. The virus genome is a linear dsDNA molecule, which varies in size within the herpesvirus family from 125 to 240 kbp (HSV: 150 kbp). The DNA is housed in the capsid, which is icosahedral, and the capsid is surrounded by the tegument. The HSV-1 tegument contains at least 15 protein species and some virus mRNA molecules. The envelope contains a large number of spikes (600--750 in HSV-1) composed of ten or more glycoprotein species. Most of the structural proteins are commonly named VP (virus protein). In HSV-1 the capsomeres are constructed from VP5: a penton is made from five molecules of VP5 and a hexon from six molecules. **HSV-1 genome organization organization** The HSV genome consists of two unique sequences each flanked by repeat sequences. The unique sequences are not of equal length: the longer is designated UL and the shorter is designated US. The HSV-1 genome encodes at least 74 proteins plus some RNAs that are not translated. Both strands of the DNA are used for coding. The inverted repeats contain some genes, so the genome contains two copies of these genes, one in each strand. **HSV-1 replication** Although HSV-1 infects only humans in nature, a variety of animal species and cell cultures can be infected in the laboratory. ***Attachment and entry*** The sequence of events at the cell surface usually involves the HSV-1 virion binding initially to heparin sulphate, and then to the main receptor. The latter can be one of several types of cell surface molecule including some nectins, which are cell adhesion molecules. The virion envelope then fuses with the plasma membrane. The nucleocapsid and the tegument proteins are released into the cytoplasm and the nucleocapsid must then be transported to the nucleus, where virus replication takes place. When the host cell is a neuron this journey is a long one. In fact, the nucleocapsid is rapidly transported along microtubules to the vicinity of a nuclear pore. The virus DNA is released into the nucleus, where the linear molecule is converted into a covalently closed circular molecule. At these sites tegument proteins play a variety of roles, including the down-regulation of host DNA, RNA and protein synthesis. One tegument protein known as virion host shutoff (VHS, a ribonuclease) protein degrades cell mRNA. Other tegument proteins are involved in the activation of virus genes, in particular the major tegument protein, VP16, which is transported to the nucleus, where it becomes associated with the virus DNA. ***Transcription and translation*** Herpesvirus genes are expressed in three phases: immediate early (IE), early (E) and late (L). **(1) The IE genes** are activated by VP16. It was noted above that VP16 from infecting virions associates with the virus DNA. It does this by binding to a complex of cell proteins including Oct-1, which binds to the sequence TAATGARAT present in the promoter of each of the IE genes. VP16 then acts as a transcription factor to recruit the host RNA polymerase II and associated initiation components to each IE gene. There are five IE proteins and all are transcription factors with roles in switching on E and L genes and in down-regulating the expression of some of these genes. **(2)** Some of **the** **E gene** products (proteins) have roles in virus DNA replication, which takes place in discrete regions of the nucleus known as replication compartments. **(3** Most of **the L gene** products are the structural proteins of the virus. ***Genome replication*** The virus DNA is replicated by E proteins. Copies of an origin-binding protein bind at one of three *ori (short for origo)* sites in the virus DNA. The protein has a helicase activity, causing the double helix to unwind at that site, and it has an affinity for the resulting single strands of DNA. The double helix is prevented from re-forming by the binding of copies of a ssDNA-binding protein. The *ori* site is then bound by a complex of three proteins, which act as a helicase, further unwinding the double helix to form a replication fork. On one of the strands a complex of the same three proteins (now acting as a primase) synthesizes a short sequence of RNA complementary to the DNA. This RNA acts as a primer in the DNA synthesis. It is thought that the circular DNA is first amplified by θ (theta) replication, and that later the replication mode switches to σ (sigma), also known as rolling circle, and the products are long DNA molecules known as concatemers, each of which consists of multiple copies of the virus genome. ***Assembly and exit of virions from the cell*** The virus envelope glycoproteins are synthesized in the rough endoplasmic reticulum and are transported to the Golgi complex. The other structural proteins, such as VP5, accumulate in the nuclear replication compartments, where procapsids are constructed. A procapsid is more rounded than a mature capsid and its structural integrity is maintained by the incorporation of scaffolding proteins. Before or during DNA packaging the scaffolding proteins are removed by a virus-encoded protease. Each procapsid acquires a genome-length of DNA, which is cut from a concatemer. Each cleavage occurs at a packaging signal at the junction of two copies of the genome. The DNA enters the procapsid via a portal at one of the vertices of the icosahedron. Once the nucleocapsid has been constructed it must acquire its tegument and envelope and then the resulting virion must be released from the cell; these are complex processes and many of the details are still unclear. It is thought that the sequence of events is as follows: **(1)** budding through the inner membrane of the nuclear envelope, giving the nucleocapsid a temporary envelope; **(2)** fusion of the temporary envelope with the outer membrane of the nuclear envelope, releasing the nucleocapsid into the cytoplasm; **(3)** acquisition of VP16 and other components of the tegument; **(4)** acquisition of the virion envelope by budding into a vesicle derived from the Golgi complex; **(5)** fusion of the vesicle membrane with the plasma membrane, releasing the virion from the cell. Although the virion envelopes are derived from membranes within the cell, virus glycoproteins are also expressed at the cell surface. These glycoproteins can cause fusion between infected cells and non-infected cells, resulting in the formation of giant cells known as syncytia. **Latent herpesvirus infection** When infection of a cell with a herpesvirus results in latency rather than a productive infection, multiple genome is switched off during latent state, but a few regions are transcribed. No virus proteins are required to maintain latency in cells that do not divide, so none are produced in neurons latently infected with HSV-1. Virus RNAs, however, are synthesized; these are known as latency-associated transcripts (LATs, non-coding RNAs). Primary transcripts are synthesized from the *LAT* gene, which is located in the terminal repeats of the genome. The LATs undergo splicing, and at least one of them plays a role in inhibiting apoptosis, thereby ensuring the survival of the neuron with its latent HSV-1 infection. EBV, in contrast, becomes latent in memory B cells, which divide from time to time; the virus therefore synthesizes proteins needed to maintain the copy number of its genome when the host cell divides. The likelihood of a latent herpesvirus infection becoming reactivated is increased if the host becomes immunocompromised; the greater the degree to which the host is immunocompromised, the greater the likelihood of reactivation. **SLIDE 12 ADENOVIRUSES** are linear, ds DNA, 30-38kbp (size varies from group to group) which has the theoretical capacity to encode 30-40 genes. Replication is divided into **EARLY** and **LATE** phases, the latter defined as beginning with the onset of DNA replication*.* Attachment to cells is rather slow, taking several hours to reach a maximum. Uptake of the adenovirus particle is a two stage process involving an initial interaction of the fiber protein with a range of cellular receptors. The penton base protein then binds to the integrin family of cell surface heterodimers allowing internalization via receptor-mediated endocytosis. **Penetration** involves phagocytosis into phagocytic vacuoles, after which the toxic activity of the pentons is responsible for rupture of the phagocytic membrane and release of the particle into the cytoplasm. Uncoating follows an ordered sequence, first the pentons, releasing a spherical, partially uncoated particle into the cytoplasm. The core migrates to the nucleus where the DNA enters through nuclear pores, whereupon it is converted into a virus DNA-cell histone complex. Before and independently of genome replication, immediate early and early mRNAs are transcribed from the input DNA. Transcription of the Adenovirus genome is regulated by virus-encoded trans-acting regulatory factors. Products of the immediate early genes regulate expression of the early genes. Multiple protein products are made from each gene by alternative splicing of mRNA transcripts. The splicing was first discovered in Adenoviruses (Philip Sharp, Nobel Prize: 1993) **SLIDE 13 PAPILLOMAVIRUSES**: Human papilloma viruses will be discussed in the lecture „Molecular Biology of Tumor Formation". **SLIDE 14 POXVIRUSES**: The viral particles (virions) are generally enveloped. They vary in their shape depending upon the species but are generally shaped like a brick or as an oval form similar to a rounded brick. The virion is exceptionally large, its size is around 200 nm in diameter and 300 nm in length and carries its genome in a single, linear, double-stranded segment of DNA. Replication of the poxvirus involves several stages. The first thing the virus does is to bind to a receptor on the host cell surface; the receptors for the poxvirus are thought to be glycosaminoglycans. After binding to the receptor, the virus enters the cell where it uncoats. Uncoating of the virus is a two step process. Firstly the outer membrane is removed as the particle enters the cell; secondly the virus particle (without the outer membrane) is uncoated further to release the core into the cytoplasm. Poxviruses are unique among DNA viruses in that they encode the majority of the enzymes required for RNA synthesis. Poxviruses are large DNA viruses that replicate entirely within the cytoplasmic compartment of the cell, and they encode their own multisubunit RNA polymerase and gene-specific transcription and termination factors. The virus-encoded RNA polymerase has sequence and structural homology to eukaryotic RNA polymerases (they stoled the RNA pol gene from the host cell). Virus-encoded and cellular proteins regulate promoter specificity by recruiting the viral RNA polymerase to one of three different classes of genes. The transcription proceeds in three phases. **(1)** During the first, early transcriptional phase, factors are expressed that are involved in viral DNA synthesis, intermediate gene expression, and modulation of the host anti-viral response. It is believed that approximately half of the poxvirus genome is transcribed during this phase, before DNA replication. **(2)** The class of genes expressed during the second or intermediate phase, immediately after DNA replication, is a much smaller group, mainly trans-activating factors for late gene transcription. **(3)** The third or late class of poxvirus genes encodes structural components of the virus, as well as components of the early transcriptional apparatus so that they can be synthesized and packaged for the next round of infection. The assembly of the virus particle occurs on the cytoskeleton of the cell. Considering the fact that this virus is large and complex, replication is relatively quick taking approximately 12 hours until the host cell dies by the release of viruses. The replication of poxvirus is unusual for a virus with double-stranded DNA because it occurs in the cytoplasm. Poxvirus encodes its own machinery for genome transcription, which makes replication in the cytoplasm possible. Most dsDNA viruses require the host cell\'s proteins to perform transcription. These host proteins are found in the nucleus, and therefore most dsDNA viruses carry out a part of their infection cycle within the host cell\'s nucleus. **SLIDES 15-16 PARVOVIRUSES** are amongst the smallest known viruses, with virions in the range 18--26 nm in diameter. They have ssDNA genome enclosed within a capsid that has icosahedral symmetry. *Dependovirus* normally replicates only when the cell is co-infected with a helper virus. Other parvoviruses that do not require helper viruses are known as autonomous parvoviruses. Not all dependoviruses have an absolute requirement for the help of an adenovirus. Other DNA viruses may sometimes act as helpers, and some dependoviruses may replicate in the absence of a helper virus under certain circumstances. Dependoviruses are valuable gene vectors. They are used to introduce genes into cell cultures for mass production of the proteins encoded by those genes, and they are being investigated as possible vectors to introduce genes into the cells of patients for the treatment of various genetic diseases and cancers. Parvoviruses have genomes composed of linear ssDNA in the size range 4--6 kb. At each end of a DNA molecule there are a number of short complementary sequences that can base pair to form a secondary structure. Some parvovirus genomes have sequences at their ends known as inverted terminal repeats (ITRs), where the sequence at one end is complementary to, and in the opposite orientation to, the sequence at the other end. The small genome of a parvovirus can encode only a few proteins, so the virus depends on its host cell (and another virus) to provide important proteins. Some of these cell proteins (a DNA polymerase and other proteins involved in DNA replication) are available only during the S phase of the cell cycle, when DNA synthesis takes place. This restricts the opportunity for parvovirus replication to the S phase. Contrast this situation with that of the large DNA viruses, such as the herpesviruses **SLIDE 17 REOVIRUSES** Icosahedral viruses with dsRNA genomes isolated from the *r*espiratory tracts and *e*nteric tracts of humans and animals, and with which no disease could be associated, became known as *reo*viruses. A large number of similar viruses have been found in mammals, birds, fish, invertebrates including insects, plants and fungi. Many of these viruses are causative agents of disease, but the original name has been preserved in the family name *Reoviridae*, and has been incorporated into the names of several of the genera within the family **SLIDES 18-21 PICORNAVIRUSES** Members of the family *Picornaviridae* are found in mammals and birds; some of the genera in the family. Picornaviruses are e.g. the Hepatitis A, Poliovirus, Rhinoviruses, Foot and mouth diseases virus. **Poliovirus was** one of the first viruses to be propagated in cell culture and was also one of the first to be plaque purified. The picornavirus genome is composed of a 7--8 kb sRNA. Covalently linked to the 5'-end of the RNA is a small protein known as VPg (virus protein, genome linked), which is involved in the regulation of transcription. By 1910, much of the world experienced a dramatic increase in polio cases and frequent epidemics became regular events, primarily in cities during the summer months. These epidemics---which left thousands of children and adults paralyzed (iron lungs)---provided the impetus for a \"Great Race\" towards the development of a vaccine. Developed in the 1950s, polio vaccines are credited with reducing the global number of polio cases per year from many hundreds of thousands to around a thousand. **SLIDES 19-27 ORTHOMYXOVIRUSES** **INFLUENZA VIRUSES** In the last 100 years there have been three major influenza pandemics\*: Spanish Flu in 1918, Asian Flu in 1957 and Hong Kong Flu in 1968. These claimed the lives of approximately 50 million, 2 million and 1 million people respectively. Added to this is the annual death toll of 250,000 to 500,000 people worldwide with a further 3 to 4 million people suffering severe illness. These statistics make influenza an extremely important pathogen. In 1997 the alarming emergence of a new, highly pathogenic subtype, H5N1 (bird flu), which has a 50% mortality rate, provided a major impetus for renewed influenza research. However the battle against influenza is going to be difficult. Recently another subtype, H1N1, has emerged. This subtype causes a relatively mild infection in humans, however is highly transmittable between people and a new influenza pandemic was declared by the World Health Organization. If this virus were to acquire some of the lethal capabilities of H5N1, then the ensuing pandemic could be devastating. **Influenza virus A** This genus has one species, influenza A virus. Wild aquatic birds are the natural hosts for a large variety of influenza A. Occasionally, viruses are transmitted to other species and may then cause devastating outbreaks in domestic poultry or give rise to human influenza pandemics. The type A viruses are the most virulent human pathogens among the three influenza types and cause the most severe disease. The influenza A virus can be subdivided into different serotypes based on the antibody response to these viruses. The serotypes that have been confirmed in humans, ordered by the number of known human pandemic deaths, are: - H1N1, which caused Spanish Flu in 1918, and Swine Flu in 2009 - H2N2, which caused Asian Flu in 1957 - H3N2, which caused Hong Kong Flu in 1968 - H5N1, which caused Bird Flu in 2004 - H7N7 - H1N2, endemic\* (local epidemic) in humans, pigs and birds - H9N2 - H7N2 - H7N3 - H10N7 **Influenza virus B** This genus has one species, influenza B virus. Influenza B almost exclusively infects humans and is less common than influenza A. This type of influenza mutates at a rate 2--3 times slower than type A and consequently is less genetically diverse, with only one influenza B serotype. As a result of this lack of antigenic diversity, a degree of immunity to influenza B is usually acquired at an early age. However, influenza B mutates enough that lasting immunity is not possible. This reduced rate of antigenic change, combined with its limited host range (inhibiting cross species antigenic shifts), ensures that pandemics of influenza B do not occur. #### Influenza virus C This genus has one species, influenza C virus, which infects humans, dogs and pigs, sometimes causing both severe illness and local epidemics. However, influenza C is less common than the other types and usually only causes mild disease in children. ### ### Structure, properties, and subtype nomenclature Influenza viruses A, B and C are very similar in overall structure. The virus particle is 80--120 nanometres in diameter and usually roughly spherical, although filamentous forms can occur. These filamentous forms are more common in influenza C, which can form cordlike structures up to 500 micrometers long on the surfaces of infected cells. However, despite these varied shapes, the viral particles of all influenza viruses are similar in composition. These are made of a viral envelope containing two main types of glycoproteins, wrapped around a central core. The central core contains the viral RNA genome and other viral proteins that package and protect this RNA. RNA tends to be single stranded but in special cases it is double. Unusually for a virus, its genome is not a single piece of nucleic acid ; instead, it contains seven or eight pieces of segmented negative-sense RNA, each piece of RNA containing either one or two genes. For example, the influenza A genome contains 11 genes on eight pieces of RNA, encoding for 11 proteins: Hemagglutinin (HA) and neuraminidase (NA) are the two large glycoproteins on the outside of the viral particles. HA is a lectin that mediates binding of the virus to target cells and entry of the viral genome into the target cell, while NA is involved in the release of progeny virus from infected cells, by cleaving sugars that bind the mature viral particles. Thus, these proteins are targets for antiviral drugs. Furthermore, they are antigenes to which antibodies can be raised. Influenza A viruses are classified into subtypes based on antibody responses to HA and NA. These different types of HA and NA form the basis of the *H* and *N* distinctions in, for example, *H5N1*. There are 16 H and 9 N subtypes known, but only H 1, 2 and 3, and N 1 and 2 are commonly found in humans. ### ### Replication Influenza infection and replication is a multi-step process: (1) firstly the virus has to bind to and enter the cell, then deliver its genome to a site where it can produce new copies of viral proteins and RNA molecules, assemble these components into new viral particles and finally exit the host cell. Influenza viruses bind through HA onto sialic acid sugars on the surfaces of epithelial cells; typically in the nose, throat and lungs of mammals and intestines of birds. After the hemagglutinin is cleaved by a protease, the cell imports the virus by endocytosis. Once inside the cell, the acidic conditions in the endosome cause two events to happen: first part of the hemagglutinin protein fuses the viral envelope with the vacuole\'s membrane, then the M2 ion channel allows protons to move through the viral envelope and acidify the core of the virus, which causes the core to dissemble and release the viral RNA and core proteins. (2) The viral RNA (vRNA) molecules, accessory proteins and RNA-dependent RNA polymerase are then released into the cytoplasm. The M2 ion channel can be blocked by amantadine drugs, preventing infection. (3a+3b) These core proteins and vRNA form a complex that is transported into the cell nucleus, where the RNA-dependent RNA polymerase begins transcribing complementary positive-sense vRNA. (4) The vRNA is either exported into the cytoplasm and translated, or remains in the nucleus (this latter one will be encapsidated). (5b) Newly synthesised viral proteins are either secreted through the Golgi apparatus onto the cell surface (in the case of NA and HA) or (5a) transported back into the nucleus to bind vRNA and form new viral genome particles. Other viral proteins have multiple actions in the host cell, including degrading cellular mRNA and using the released nucleotides for vRNA synthesis and also inhibiting translation of host-cell mRNAs. Negative-sense vRNAs that form the genomes of future viruses, RNA-dependent RNA polymerase, and other viral proteins are assembled into a virion. HA and NA molecules cluster into a bulge in the cell membrane. (6) The vRNA and viral core proteins leave the nucleus and enter this membrane protrusion. (7) The mature virus buds off from the cell in a sphere of host phospholipid membrane, acquiring HA and NA with this membrane coat. As before, the viruses adhere to the cell through HA; the mature viruses detach once their NA has cleaved sialic acid residues from the host cell. Drugs that inhibit neuraminidase, such as oseltamivir, therefore prevent the release of new infectious viruses and halt viral replication. After the release of new influenza viruses, the host cell dies. Because of the absence of RNA proofreading enzymes, the RNA-dependent RNA polymerase that copies the viral genome makes an error roughly every 10 thousand nucleotides, which is the approximate length of the influenza vRNA. Hence, the majority of newly manufactured influenza viruses are mutants; this causes \"antigenic drift\", which is a slow change in the antigens on the viral surface over time. The separation of the genome into eight separate segments of vRNA allows mixing or reassortment of vRNAs if more than one type of influenza virus infects a single cell. The resulting rapid change in viral genetics produces antigenic shifts, which are sudden changes from one antigen to another. These sudden large changes allow the virus to infect new host species and quickly overcome protective immunity, which is important in the emergence of pandemics. ### ***New strains of INFLUENZA A*** From time to time a virus emerges with a new combination of HA and NA genes formed by reassortment, and causes a pandemic. The hosts of influenza A viruses are principally birds that frequent aquatic habitats. The birds (e.g. ducks, geese, gulls) acquire infections by ingestion or inhalation and the viruses infect their intestinal and/or respiratory tracts. Infection with most virus strains results in few or no signs of disease, but some strains are highly pathogenic and can kill their avian hosts. The viruses can be spread to new areas when the birds migrate. Some influenza A viruses infect mammalian species including pigs, horses and humans; the respiratory tract is the main site of virus replication. Normally humans are infected only with viruses that have H type 1, 2 or 3 and N type 1 or 2. Patients are commonly very ill and some die, either as a direct result of the virus infection, or indirectly from secondary pathogens, which are able to infect as a result of damage to the respiratory epithelium. Some avian strains of influenza A virus can be highly pathogenic in birds, can be transmitted from wild birds to domestic poultry and can be transmitted to humans. This was the situation in Hong Kong in 1997, when an H5N1 virus caused an outbreak of serious disease in poultry. H5N1 viruses appeared in a number of Asian countries in 2003, in Europe in 2005 and in Africa in 2006. Millions of ducks, chickens and turkeys died from the disease or were slaughtered; H5N1 viruses also infected humans, causing severe respiratory disease, culminating in death in many cases. At about the same time H9N2 viruses emerged in Asia, causing disease in poultry with some transmission to humans. Most, if not all, of the human cases of 'bird flu' caused by H5N1 and H9N2 viruses were in people who work with poultry and who presumably became infected as a result of direct contact with virus on the birds themselves, or in their faeces. These avian influenza viruses appeared to have little or no propensity for human-to-human transmission. **SLIDES 28, 29 RHABDOVIRUSES** The rhabdoviruses have minus-strand RNA genomes in the size range 11--15 kb. The name of these viruses is derived from the Greek word *rhabdos*, which means a rod. The virions of some rhabdoviruses, especially those infecting plants, are in the shape of rods with rounded ends, while others, especially those infecting animals, are bullet shaped. Rhabdoviruses are found in a wide range of hosts, including mammals, fish, plants and insects. Many rhabdoviruses have very wide host ranges and replicate in the cells of diverse types of host, especially the so-called 'plant' rhabdoviruses, which replicate in their insect vectors as well as in their plant hosts. ***Rabies virus***, like many rhabdoviruses, has an exceptionally wide host range. Infection with rabies virus normally occurs as a result of virus in saliva gaining access to neurons through damaged skin. The infection spreads to other neurons in the central nervous system, then to cells in the salivary glands, where infectious virus is shed into the saliva. The rhabdovirus virion is an enveloped, rod- or bullet-shaped structure containing five protein species. The nucleoprotein (N) coats the RNA at the rate of one monomer of protein to nine nucleotides, forming a nucleocapsid with helical symmetry. Associated with the nucleocapsid are copies of P (phosphoprotein) and L (large) protein. The L protein is well named, its gene taking up about half of the genome. Its large size is justified by the fact that it is a multifunctional protein. The M (matrix) protein forms a layer between the nucleocapsid and the envelope, and trimers of G (glycoprotein) form spikes that protrude from the envelope. **SLIDES 30-46 RETROVIRUSES**: The retroviruses are RNA viruses that copy their genomes into DNA during their replication. Until the discovery of these viruses it had been dogma that the transfer of genetic information always occurs in the direction of DNA to RNA, so finding that some viruses carry out 'transcription backwards' (reverse transcription) caused something of a revolution. We now know that reverse transcription is carried out, not only by these RNA viruses, but also by some DNA viruses (Hepadnaviruses) and by uninfected cells (by the retrotransposons). Many retroviruses can cause cancer in their hosts (HIV does it very rarely and by only chance; will be discussed in lecture "Molecular Biology of Tumor Formation"). The virion contains two copies of the RNA genome; hence the virion can be described as diploid. The two molecules are present as a dimer, formed by base pairing between complementary sequences. As well as the virus RNA, the virion also contains molecules of host cell RNA that were packaged during assembly. This host RNA includes a molecule of transfer RNA (tRNA) bound to each copy of the virus RNA through base pairing. The sequence in the virus RNA that binds a tRNA is known as the primer binding site. Each retrovirus binds a specific tRNA. A number of protein species are associated with the RNA. The most abundant protein is the nucleocapsid (NC) protein, which coats the RNA, while other proteins, present in much smaller amounts, have enzyme activities. Encasing the RNA and its associated proteins is the capsid, which appears to be constructed from a lattice of capsid (CA) protein. The shape of the capsid can be spherical, cylindrical or conical depending on the virus. A layer of matrix (MA) protein lies between the capsid and the envelope. Associated with the envelope are two proteins: a transmembrane (TM) protein bound to a heavily glycosylated surface (SU) protein. The genes encoding the virus proteins are organized in three major regions of the genome: ***gag*** (*g*roup-specific *a*nti*g*en) -- internal structural proteins ***pol*** (*pol*ymerase) -- enzymes ***env*** (*env*elope) -- envelope proteins **Human Immunodeficiency Virus (HIV)** belongs to the family of Retroviridae and genus of lentivirus. There are two types of human immunodeficiency virus (HIV-1 and HIV-2), which each evolved from a different simian immunodeficiency virus (SIV). Both viruses emerged in the late 20th century. In contrast to the SIVs, which appear not to harm their natural primate hosts, HIV infection damages the immune system, leaving the body susceptible to infection with a wide range of pathogens. This condition is called acquired immune deficiency syndrome (**AIDS**). HIV-1 is much more prevalent than HIV-2; it is HIV-1 that is largely responsible for the AIDS pandemic, while HIV-2 is mainly restricted to West Africa. Now, in each year of the early 21st century there are approximately 5 million new HIV infections, and approximately 3 million deaths from AIDS, which has become the fourth biggest cause of mortality in the world. The magnitude of this problem has resulted in the allocation of huge resources to the study of these viruses, major objectives being the development of anti-viral drugs and a vaccine. So far there has been qualified success in achieving the first of these objectives. HIV-1 and HIV-2 have genomes about 9.3 kb in length. The genomes encode auxiliary genes in addition to *gag*, *pol* and *env*, and so the viruses are classed as complex retroviruses. The auxiliary genes have many roles in controlling virus gene expression, transporting virus components within the cell and modifying the host's immune response. Some of the auxiliary gene products have multiple roles. All three reading frames are used and there is extensive overlapping; e.g., part of *vpu* in frame 2 overlaps *env* in frame 3. The sequences for *tat* and *rev* are split, the functional sequences being formed when the transcripts are spliced. HIV-2 has similar genes to HIV-1, except that it has no *vpu* gene, but it has a *vpx* gene which is related to *vpr*. ***Attachment and entry*** The cell receptor for HIV-1 is [CD4], which is found on several cell types, including helper T cells and some macrophages; [CD4 T cells] are the main target cells. Attachment of the virion occurs when a site on [gp120] recognizes a site on the outer domain of CD4. As well as binding to receptor molecules an HIV-1 virion must also attach to a co-receptor on the cell surface. The molecules that act as co-receptors have seven transmembrane domains and are [chemokine receptors]. During immune responses they bind chemokines and these interactions control leukocyte trafficking and T cell differentiation. Most chemokines fall into one of two major classes, determined by the arrangement of cysteine residues near the N terminus: C--C and C--X--C, where C = cysteine and X = any amino acid. The chemokine receptors are designated CCR and CXCR, respectively. A number of these molecules on T cells act as co-receptors for HIV-1, particularly CCR5 and CXCR4. Most HIV-1 strains use CCR5 and are known as **R5 strains**. It is interesting to note that in some individuals who have had multiple exposures to the virus, but have not become infected, there is a 32-nucleotide deletion in the *CCR5* gene. Individuals who are homozygous for this mutation express no CCR5 on their cells and are highly resistant to infection with HIV-1, while those who are heterozygous have increased resistance. The mutation is found mainly in Europeans. HIV-1 strains that use CXCR4 as a co-receptor are known as **X4 strains**, and there are some strains (R5X4 strains) that can use either co-receptor. R5 strains do not infect na¨ıve T cells, but all three strains infect memory T cells. The interaction of gp120 with the receptor and co-receptor results in a dramatic re-arrangement of gp41, which proceeds to fuse the membranes of the virion and the cell. The contents of the virion envelope are released into the cytoplasm and develop into the reverse transcription complex, which contains the MA, Vpr, RT and IN proteins, as well as the virus genome. ***Reverse transcription and transport to the nucleus*** The reverse transcription complex associates rapidly with microtubules. Reverse transcription is primed by tRNAlys-3. After reverse transcription has been completed, the pre-integration complex, which contains host proteins as well as virus proteins, is moved along microtubules towards the nucleus. Most retroviruses can productively infect only if there is breakdown of the nuclear membranes. The preintegration complex of HIV, however, can enter an intact nucleus, such as that of a resting T cell or a macrophage, and is presumably transported through a nuclear pore. ***Early gene expression*** Transcription is initiated after cell transcription factors bind to promoter and enhancer sequences in the upstream LTR (long terminal repeat). Transcription is terminated in the downstream LTR. The largest RNAs are genome length (about 9.3 kb), while the other two size classes are each made up of a number of mRNA species that have undergone splicing; mRNAs that have been spliced once are around 4.5 kb, while mRNAs that have undergone two or more splicing events ('multiply spliced' transcripts) are around 2 kb. The virus genome has a number of splice donor and acceptor sites; these enable splicing events that result in more than 30 mRNA species. Early in infection most of the primary transcripts are multiply spliced and these RNAs are translated into the Nef, Tat and Rev proteins. The **Nef** (Negative regulatory factor) protein acquired its name because it was originally thought to have an inhibitory effect on HIV replication, though later work showed that this protein stimulates replication! In infected cells Nef alters the endosome trafficking pathway, reducing expression at the cell surface of CD4 and MHC class I and II proteins. These changes can shield HIV-infected cells from immune surveillance. The **Tat** (***T*** rans***a***ctivator of ***t***ranscription) protein plays an important role in enhancing transcription. A nuclear localization signal directs Tat to the nucleus, where it binds to a sequence at the 5' end of nascent virus transcripts: this sequence is known as the transactivation response (TAR) element. Cell proteins also bind to [TAR], and among these proteins is a kinase, which phosphorylates components of the RNA polymerase complex. Phosphorylation increases the processivity of the enzyme along the proviral template. Tat therefore functions as a transcription factor, but an unusual one, in that it binds not to DNA, but to RNA. In the absence of Tat most transcripts are incomplete, though early in infection sufficient are completed to allow the synthesis of a small amount of Tat, which then significantly boosts the synthesis of genome-length RNA. The other early protein, the **Rev** (***R***egulator of ***e***xpression of ***v***irion proteins) protein, has a nuclear localization signal. As Rev accumulates in the nucleus it causes a shift from early to late protein synthesis by binding to the Rev response element ([RRE]) in the virus RNA. ***Late gene expression*** The **Gag** and **Gag--Pol** are translated from unspliced transcripts, with Gag--Pol translated when a ribosomal frameshift takes place. This occurs on roughly five per cent of occasions when a ribosome traverses the sequence UUUUUUA. This sequence (known as a slippery sequence), together with a downstream secondary structure, causes the ribosome to slip from reading frame 1 to reading frame 3. After the frameshift has taken place translation continues through the *pol* region, yielding the Gag--Pol polyprotein. The remaining virus proteins (Vif, Vpr, Vpu and Env) are translated from singly spliced transcripts. Vpu and Env are translated in the rough endoplasmic reticulum from a bicistronic transcript. Env becomes heavily glycosylated. Trimers of Env are formed before the molecules are cleaved to form the envelope proteins gp120 and gp41; the cleavage is carried out by furin, a host protease located in the Golgi complex. Vpu is a membrane-associated protein and is required for efficient budding of virions from the plasma membrane. ***Assembly and exit of virions*** Formation of the RNA dimer that will constitute the genome of a new virion commences by base pairing between complementary sequences in the loop of the dimerization initiation site near the 5' end of each RNA. Molecules of Gag and Gag--Pol form an orderly arrangement, and their domains bind to the virus genome and to other proteins that will become incorporated into the virion. Gag--Pol dimers are formed; these undergo self cleavage to form the virus enzymes, including the protease, which is a dimer. The protease then cleaves the Gag polyproteins into the constituents of the mature virion. **Variability of the HIV genome** The extensive genetic variability of HIV is primarily due to the high error rates of the viral reverse transcriptase, which results in approximately 10 genomic base changes per replication cycle. In addition to substitutions, deletions and insertions also occur, although the frequency of these genetic errors is more difficult to estimate. The envelope gene (env) seems to be subject to the most extensive genetic variation, although alterations also occur in other genes. HIV-1 undergoes continuous genetic variability within individual patients, who usually harbor a swarm of highly related but individually distinguishable viral variants, which are referred to as quasispecies, with a heterogeneity usually not exceeding 2-5% in the env gene. In addition, recent data have provided evidence that genomic recombination between two different HIV-1 populations frequently occurs in vivo, resulting in biologically viable viruses with mosaic genomes, a phenomenon which may result in additional HIV genetic variability and viral genetic shifts. **The origin of HIV** **HIV-1** When a viral transfer between animals and humans takes place, it is known as zoonosis. Below are some of the most common theories about how this \'zoonosis\' took place, and how SIV became HIV in humans: **(1) The \'hunter\' theory** The most commonly accepted theory is that of the \'hunter\'. In this scenario, SIV was transferred to humans as a result of chimps being killed and their blood getting into cuts or wounds on the hunter. #### #### (2) The oral polio vaccine theory Some other rather controversial theories have contended that HIV was transferred iatrogenically (i.e. via medical interventions). One particularly well-publicized idea is that polio vaccines played a role in the transfer. Live polio vaccine was cultivated in cell culture derived from macaque kidney cells. It was supposed that the donor monkey might have been infected with SIV. However, analysis of stores a phial of polio vaccine revealed neither SIV nor HIV in the cells. #### **HIV-2** HIV-2 is thought to come from the SIV in Sooty Mangabeys rather than chimpanzees, but the crossover to humans is believed to have happened in a similar way. It is far rarer, significantly less infectious and progresses more slowly to AIDS than HIV-1. As a result, it infects far fewer people, and is mainly confined to a few countries in West Africa. **Nobel Prizes** Howard Temin was awarded a Nobel Prize for their provirus hypothesis of retroviruses. The story of the other Nobel Prize is as follows. One of the tackiest sagas in the history of medicine unfolded in the decade after HIV was discovered in 1983. On the face of it, celebrations were in order because it had taken scientists just two years to discover what was causing AIDS after the first cases emerged in 1981. Instead, the world\'s public were treated to an interminable squabble between two teams - one in France and one in the US - over who actually discovered the virus, whose test for the virus was patented first, and whether one team had \"appropriated\" viral samples from the other. One team, at the National Cancer Institute in Bethesda, Maryland, was led by Robert Gallo. The other, at the Pasteur Institute in Paris, was led by Luc Montagnier. Now, the whole saga has been raked up again because Montagnier and his colleague Françoise Barré-Sinoussi awarded the prize in 2008. **SLIDES 47, 48 HEPADNAVIRUSES** The hepadnaviruses got their name because they cause ***hepa***titis and they have ***DNA*** genomes. They are known as hepatitis B viruses (HBVs). Some members infect mammals and some infect birds. The best-known hepadnavirus is that which infects humans; it is commonly referred to as **HBV**, and is of major importance as an agent of disease and death. The hepadnaviruses are especially fascinating for two reasons. (1) First they have very small genomes, which are used with great economy to encode the virus proteins and to control expression of the virus genes. (2) Second, their DNA genomes are replicated via an RNA intermediate. In other words, their replication involves reverse transcription, so they are very different from DNA viruses that replicate their DNA directly to DNA. No-one knows how many people are infected with HBV, but the figure is probably around 400 million. The majority is in Asia and many are in Africa. Virus is present in the blood and semen of infected individuals and the modes of transmission generally parallel those for HIV transmission. There are over 50 million new HBV infections each year, the majority in babies who acquire the infection from their mothers. Over 8 million infections per year are thought to result from the re-use of syringes and needles for injections, mainly in the developing world. Many HBV infections result in mild symptoms or are asymptomatic, especially in children. It is in children, however, that HBV infection is most likely to become persistent, with 90--95 per cent of those infected as newborn infants becoming long-term carriers, compared with 1--10 per cent of those becoming infected as adults. Individuals who are persistently infected with HBV may remain healthy for much of the time, but some develop severe hepatitis, which may lead to cirrhosis and eventually to liver cancer. These diseases resulting from HBV infection cause about one million deaths per year. At 3.2 kb, the HBV genome is very small, though there are viruses with smaller genomes. There are four ORFs, from which seven proteins are translated, so a large amount of coding information is packed into the small genome. The virus cleverly achieves this by using every nucleotide in the genome for protein coding and by reading more than half of the genome in two reading frames. The P ORF, which occupies about 80 per cent of the genome, overlaps the C and X ORFs and the entire S ORF is within the P ORF. A further way in which the virus maximizes its coding capacity is by expressing the L protein in two different conformations that have different functions. In one conformation L protein molecules act as virus attachment proteins, while in the other they bind the virion envelope to the capsid. As the entire genome is involved in coding for protein, it follows that all the regulatory sequences, such as promoters, are within protein coding sequences. The genome contains direct repeats of 11 nucleotides known as DR1 and DR2. Expression of the pre-S1--pre-S2--S region gives rise to three proteins. Translation of the S region produces the S protein, translation of pre-S2--S produces the M protein and translation of the complete ORF produces the L protein. Similarly, expression of the pre-C--C region gives rise to two proteins. Even though the virus maximizes the use of its small genome it encodes only seven proteins, so it is heavily dependent on host cell functions. **Emerging viruses** **NO SLIDE** The term 'emerging virus' is used in a number of contexts: it may refer to a virus that has recently made its presence felt by infecting a new host species, by appearing in a new area of the world or by both. Sometimes a virus is described as a 're-emerging virus' if it has started to become more common after it was becoming rare. Foot and mouth disease virus re-emerges in the UK from time to time. Human activities may increase the likelihood of virus emergence and re-emergence. Other activities that may result in virus emergence involve close contact with animals, including the hunting and killing of non-human primates for bush meat. It has been shown that viruses such as simian immunodeficiency viruses (SIVs) can be present in the meat and there is a risk of acquiring an infection when the meat is handled. Simian-to-human transmission of SIVs is thought to have occurred several times, resulting in the major groups of HIV-1 and HIV-2, and there is concern that further viruses might emerge as a result of contact with bush meat. If a virus jumps into a new host species it may undergo some evolutionary changes in the new host, resulting in a new virus. This is how HIV-1 and HIV-2 were derived from their SIV precursors. Other new viruses emerge when recombination and reassortment result in new viable combinations of genes; new strains of influenza A virus come into this category. ***FILOVIRUSES*** In 1967 in the town of Marburg in Germany some laboratory workers became ill with a haemorrhagic fever. These people had been in contact with blood, organs and cell cultures from African green monkeys caught in Uganda. Investigations revealed that the monkeys had been infected with a virus that had been transmitted to the laboratory staff and from them to the hospital staff. It was named **Marburg virus** and is now classified in the family *Filoviridae*, named from the Latin *filum*, meaning a thread. In 1976 there were outbreaks of a similar disease in Africa near the River Ebola in the Democratic Republic of Congo and in Sudan, which was caused by the **Ebola virus**. Since then there have been a number of outbreaks of disease caused by Ebola and Marburg viruses across central Africa, from Ivory Coast in the west to Kenya in the east and to Angola in the south. The way in which the outbreaks start has long been a mystery. There is increasing evidence that some outbreaks start when a human becomes infected as a result of contact with the blood of an infected non-human primate. It has since been found that gorillas and chimpanzees can be infected with Ebola virus, which may be responsible for significant mortality of these species. Ebola and Marburg viruses are present in the blood of infected hosts and transmission to humans can occur through contact with the flesh of infected animals after they have been hunted and killed. Human-to-human transmission readily occurs through contact with the blood of infected individuals. There are several species of Ebola virus in Africa. A further species has appeared in the US and Italy in monkeys imported from the Philippines; there is serological evidence that animal handlers have been infected, but there have been no reports of this virus causing disease. It is thought likely that additional animal species act as 'reservoirs' of Marburg and Ebola viruses, and that infection in these species is likely to cause few or no signs of disease. There have been many expeditions to search for reservoir hosts, but very few produced positive results. There remain unanswered questions concerning reservoir species and host ranges of these viruses. ***SARS CORONAVIRUS*** In 2002 a new human respiratory disease emerged in southern China. The following year one of the doctors who had been treating patients travelled to Hong Kong, where he became ill and died. Subsequently, people who had stayed in the same hotel as the doctor travelled to Singapore, Vietnam, Canada and the US, taking the infectious agent with them. The epidemic of severe acute respiratory syndrome (SARS) was under way. The signs and symptoms of SARS resemble those of influenza. About 90 per cent of patients recovered, but for the remainder the infection proved to be lethal. These were mainly individuals who had an underlying condition such as diabetes, heart disease or a weakened immune system. On the face of it SARS is a respiratory tract disease, but in many patients the infection spread to other parts of the body. The causative agent was found to be a new coronavirus. No natural reservoir for the SARS coronavirus has been found. The SARS outbreak was brought under control by quarantine measures, but only after there had been over 8000 cases with nearly 800 deaths. ***WEST NILE VIRUS*** In 1999 some people in New York became ill with apparent viral encephalitis; several of them died. At about the same time several birds in the zoo became ill, and deaths of wild birds, especially crows, were reported. Diagnostic tests revealed that these human patients and the birds were infected with West Nile virus (WNV). **Reemerging viruses** Measles and mumps viruses are members of the family *Paramyxoviridae*. Both are important human pathogens, especially measles, which is a major cause of mortality in developing countries. Until the late 1990s cases of measles and mumps had been declining in the UK as a result of widespread uptake of the measles, mumps and rubella (MMR) vaccine, but after fears were raised surrounding the safety of the vaccine fewer children were vaccinated. Measles, and especially mumps, began to 'reemerge', with hundreds of measles cases and thousands of mumps cases each year. **Bacteriophages** **SLIDE 49** This topic will be discussed at the lecture "Bacteria" **Prions** **SLIDE 53 Prions** (not virus!) The causative agents of prion are protein molecules from within the cells of the host; no nucleic acid has been found associated with them. The infectious agent of prion is misfolded prion protein, then transmission of a prion disease implies that the introduction of the misfolded protein into the body of a new host initiates the misfolding of protein molecules in that host. If the agent is 'transmitted' to other species then, because each species makes a specific prion protein, the molecules that become misfolded have the amino acid sequence of the recipient prion, not that of the donor. The protein-only hypothesis suggests that the agent is derived from one of the body's own proteins, and this can explain the inherited forms of the disease. According to the hypothesis, these diseases develop as a consequence of the inheritance of a gene encoding a prion protein with an amino acid sequence that has a high probability of misfolding. There remain many controversies and there is still much to be learnt about the prion disease and their causative agents. Progress in research is slow as a result of the long incubation periods of the diseases.