Torovirus- An Overview

Torovirus is a genus of viruses that belong to the order Nidovirales, the family Coronaviridae, and the subfamily Torovirinae . The name torovirus comes from the Latin word "torus", meaning a ring or a doughnut, because of the distinctive shape of the virus particles. Torovirus is the only genus in the monotypic subfamily Torovirinae, and it also contains only one subgenus, Renitovirus .

There are four recognized species of torovirus: bovine torovirus (BToV), equine torovirus (EToV), porcine torovirus (PToV), and human torovirus (HToV) . These viruses primarily infect vertebrates, especially cattle, pigs, horses, and humans, and are associated with gastroenteritis and diarrhea . Among the four species, there is little genetic divergence (20-40%), and antigenic cross-reactivity has been observed between EToV, BToV, and HToV .

The first torovirus was discovered in 1972 from a rectal swab of a horse with diarrhea and was named Berne virus (later renamed as EToV) . EToV is the only torovirus that can be grown in cell culture, using equine kidney or dermis cells . In 1979, another torovirus was isolated from an outbreak of neonatal gastroenteritis in calves from a dairy farm in Breda, Iowa, and was named Breda virus (later renamed as BToV) . In 1984, torovirus-like particles were detected by electron microscopy in human fecal samples from patients with gastroenteritis and were named human toroviruses (HToVs) . Porcine toroviruses (PToVs) were identified later in 1998 from pigs with diarrhea.

Toroviruses are classified as group IV viruses according to the Baltimore classification system, which is based on the nature and polarity of the viral genome. Toroviruses have a single-stranded RNA genome with positive polarity, meaning that it can act as a messenger RNA and be directly translated by the host cell ribosomes . The genome is about 25-30 kilobases long and has a 5` cap and a 3` polyadenylated tail . The genome contains six open reading frames (ORFs) that encode for the replicase proteins (ORF1a and ORF1b) and the structural proteins (ORF2 to ORF5), which are spike (S), membrane (M), hemagglutinin-esterase (HE), and nucleocapsid (N) proteins .

Toroviruses are enveloped viruses with a lipid bilayer derived from the host cell membrane . The envelope contains three types of glycoproteins: S, M, and HE . The S protein forms club-shaped spikes on the surface of the virus that mediate attachment and fusion with the host cell receptors . The M protein is the most abundant protein in the envelope and plays a role in virus assembly and budding . The HE protein is a receptor-destroying enzyme that cleaves sialic acid residues from glycoproteins and glycolipids on the host cell surface and prevents virus aggregation . The N protein is the major component of the nucleocapsid, which is a helical structure that encloses the genomic RNA . The nucleocapsid has a unique C-shape or open torus morphology that gives the virus its name .

Torovirus is a genus of viruses in the order Nidovirales, in the family Coronaviridae, in the subfamily Torovirinae. Toroviruses are pleomorphic, enveloped particles, 120–140 nm in diameter. The virus particle has surface spike proteins that are club-shaped 15 to 20 nm in length and are evenly dispersed over the surface. A nucleocapsid is doughnut-shaped with helical symmetry. The tubular nucleocapsid is bent into an open torus, hence the name “torovirus”.

The molecular weights of the nucleocapsid protein is 19 kDa. These are enveloped viruses with non-segmented and positive-sense (single stranded) RNA genome of 20 to 25 kilobases. Virions contain the glycoproteins spikes (S), membrane protein (M), a core nucleoprotein (N) and hemagglutinin-esterase (HE). Toroviruses have a buoyant density of 1.14–1.18 g/cm3 in sucrose. Virus infectivity is stable between pH 2.5 and 9.7 but rapidly inactivated by heat, organic solvents, and irradiation.

Torovirus shares some common characteristics with members of the related family Coronaviridae as they are round, pleomorphic, enveloped viruses about 120 to 140 nm in diameter .

The genome of torovirus is a single-stranded, positive-sense RNA molecule of about 20 kilobases (kb) in length. It is the largest RNA genome among the animal viruses. The genome has a 5` cap and a 3` poly(A) tail, which are essential for translation and stability. The genome contains seven open reading frames (ORFs) that encode for both structural and non-structural proteins.

The first two ORFs, ORF1a and ORF1b, occupy about two-thirds of the genome and are translated from the genomic RNA. They encode for the viral replicase complex, which consists of 16 non-structural proteins (nsp1 to nsp16). These proteins are involved in RNA synthesis, modification, processing and capping. ORF1b is translated by a -1 ribosomal frameshift mechanism that occurs at a conserved slippery sequence near the end of ORF1a.

The remaining five ORFs, ORF2 to ORF6, are translated from a set of subgenomic RNAs (sgRNAs) that are produced by discontinuous transcription during negative-strand RNA synthesis. These ORFs encode for the structural proteins of the virus: the spike glycoprotein (S), the envelope protein (E), the membrane glycoprotein (M), the nucleocapsid protein (N) and the hemagglutinin-esterase protein (HE). The S protein forms the characteristic club-shaped spikes on the surface of the virion and mediates attachment and fusion with host cells. The E protein is a small integral membrane protein that may play a role in virus assembly and release. The M protein is the most abundant structural protein and forms the matrix layer under the viral envelope. The N protein binds to the genomic RNA and forms the helical nucleocapsid. The HE protein is a type I membrane glycoprotein that has both hemagglutinin and acetylesterase activities. It is involved in receptor binding and virus entry.

The torovirus genome has a high degree of genetic diversity and recombination, which may contribute to its adaptation and evolution. The torovirus genome also shows some similarities and differences with other members of the Nidovirales order, such as coronaviruses and arteriviruses. For example, toroviruses share a common gene order and some conserved motifs with coronaviruses, but they have a unique HE gene that is absent in coronaviruses. Toroviruses also have a shorter genome and fewer ORFs than coronaviruses. On the other hand, toroviruses have more sgRNAs and a larger N protein than arteriviruses.

The torovirus genome is an important target for molecular diagnosis, epidemiology, phylogeny and vaccine development. Several molecular methods have been developed to detect and characterize torovirus genomes, such as reverse transcription-polymerase chain reaction (RT-PCR), real-time RT-PCR, nested RT-PCR, multiplex RT-PCR and sequencing. These methods can identify different torovirus species, strains and genotypes based on their genomic variations.

• The geographic distribution of torovirus in human and animals is worldwide, with the virus identified in countries such as Brazil, Belgium, Canada, Costa Rica, France, Germany, Great Britain, Hungary, India, The Netherlands, New Zealand, South Africa, the United States, and most recently Austria.
• Toroviruses have been detected in stools of children and adults with diarrhea in developed countries. However, in these studies there was no epidemiologic association with illness, and the detections could not be confirmed using additional tests.
• The virus, originally designated Breda virus, was first isolated during an outbreak of severe neonatal gastroenteritis with 56.5% morbidity and 8.7% mortality in cattle from dairy farms round the township Breda, Iowa, and duly identified as the etiological agent.
• Porcine torovirus (PToV) is a potential enteric swine pathogen, found at especially high rates in piglets with diarrhea. It was first reported in the Netherlands in 1998 and has emerged in many countries around the world.
• Infections are generally asymptomatic and have not directly caused large economic losses, though co-infections with other swine pathogens and intertype recombination may lead to unpredictable outcomes.
• Antigenic cross-reactivity has revealed a relationship between equine torovirus (EToV), bovine torovirus (BToV), and human torovirus (HToV). Among them there is little genetic divergence (20–40%).

The life cycle of toroviruses involves replication within the infected host cell. These viruses predominantly bud into the lumen of the Golgi cisternae in the cytoplasm. The replication process can be summarized as follows:

• Attachment of the viral spike (S) protein or hemagglutinin-esterase (HE) protein to host receptors mediates endocytosis of the virus into the host cell. Endocytosis can occur via clathrin-mediated endocytosis, caveolin-mediated endocytosis or clathrin- and caveolin-independent endocytosis.
• Fusion of virus membrane with the endosomal membrane, releasing ssRNA(+) genome into the cytoplasm.
• Synthesis and proteolytic cleavage of the replicase polyprotein occurs. The replicase polyprotein consists of two open reading frames (ORF1a and ORF1b) that are translated from genomic RNA and constitute the viral replicase enzymes. ORF1b is translated by ribosomal frameshifting. The resulting proteins pp1a and pp1ab are processed into the viral polymerase (RdRp) and other non-structural proteins involved in RNA synthesis.
• Replication occurs in viral factories where a dsRNA genome is synthesized from the genomic ssRNA(+). The replication occurs in membranous invaginations of the rough endoplasmic reticulum (REG), possibly to avoid dsRNA intermediate detection.
• The dsRNA genome is transcribed/replicated thereby providing viral mRNAs/new ssRNA(+) genomes. Each RNA (genomic and subgenomic) is translated to yield only the protein encoded by the 5′-most ORF.
• Synthesis of structural proteins encoded by subgenomic mRNAs. These include the S, M, N and HE proteins that are essential for virion assembly and infectivity.
• Release of new virion by budding at endoplasmic reticulum (ER) or Golgi apparatus which implies that the viral particle are exported by cellular exocytosis.

Toroviruses are enteric viruses that infect the epithelial cells of the small and large intestine, causing diarrhea and gastroenteritis in humans and animals. The pathogenesis of torovirus infection is not fully understood, but some general steps can be summarized as follows:

• Transmission: Toroviruses are transmitted by the fecal-oral route, through contact with contaminated feces, food, water, or fomites. They can also spread by respiratory droplets or aerosols from infected individuals.
• Attachment and entry: Toroviruses attach to the host cell receptors by their spike (S) proteins or hemagglutinin-esterase (HE) proteins, which mediate endocytosis of the virus into the cell. Endocytosis can occur via different pathways, such as clathrin-mediated, caveolin-mediated, or clathrin- and caveolin-independent endocytosis.
• Replication and transcription: Toroviruses have a positive-sense single-stranded RNA genome of about 20-30 kb, which serves as both the genome and the viral messenger RNA. The genome contains six open reading frames (ORFs), which encode the replicase (ORF1a and ORF1b) and the structural proteins (S, M, HE, and N). The replicase is translated from the genomic RNA and processed into the viral polymerase and other non-structural proteins involved in RNA synthesis. The structural proteins are translated from subgenomic RNAs, which are produced by discontinuous transcription from a negative-sense RNA intermediate. The replication and transcription occur in membranous invaginations of the endoplasmic reticulum or Golgi apparatus, where the virus forms replication complexes that avoid detection by the host immune system.
• Assembly and release: Toroviruses assemble their nucleocapsids by wrapping the genomic RNA with the nucleocapsid (N) protein, forming a doughnut-shaped structure with helical symmetry. The nucleocapsids then acquire the envelope by budding at the endoplasmic reticulum or Golgi apparatus, where they incorporate the spike (S), membrane (M), and hemagglutinin-esterase (HE) proteins. The enveloped virions are then released by exocytosis or cell lysis.
• Cellular damage and immune response: Toroviruses cause cellular damage by inducing apoptosis, necrosis, or cytopathic effects in the infected cells. They also trigger an innate and adaptive immune response in the host, involving cytokines, chemokines, interferons, natural killer cells, macrophages, dendritic cells, B cells, and T cells. However, toroviruses can evade or modulate the immune response by various mechanisms, such as antigenic variation, interference with interferon signaling, inhibition of apoptosis, suppression of cytokine production, or induction of immunopathology.

The pathogenesis of torovirus infection may vary depending on the host species, age, immune status, viral strain, co-infection with other pathogens, or environmental factors. Some of the factors that influence the severity and outcome of torovirus infection are:

• Host species: Toroviruses have a broad host range and can infect humans and animals such as cattle, horses, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, ferrets, monkeys, camels, deer, antelopes, giraffes, and elephants. However, not all toroviruses cause disease in all hosts. For example, equine torovirus (EToV) is apathogenic in horses but can cause diarrhea in calves; bovine torovirus (BToV) is pathogenic in calves but not in adult cattle; human torovirus (HToV) is associated with gastroenteritis in children but not in adults; porcine torovirus (PToV) is pathogenic in pigs but not in other animals.
• Age: Toroviruses are more likely to cause severe disease in young animals than in older ones. This may be due to differences in intestinal development, microbiota composition, mucosal immunity, or susceptibility to co-infections. For example, BToV causes neonatal calf diarrhea with high morbidity and mortality; EToV causes diarrhea in young calves but not in older ones; HToV causes gastroenteritis in infants and children but not in adults; PToV causes diarrhea in piglets but not in adult pigs.
• Immune status: Toroviruses can cause more severe disease in immunocompromised hosts than in immunocompetent ones. This may be due to impaired innate or adaptive immune responses that fail to control viral replication or inflammation. For example, HToV can cause nosocomial infections in infants with necrotizing enterocolitis or other underlying conditions; BToV can cause diarrhea in calves with concurrent infections with rotavirus or coronavirus; PToV can cause diarrhea in piglets with concurrent infections with transmissible gastroenteritis virus or porcine epidemic diarrhea virus.
• Viral strain: Toroviruses have a high genetic diversity and can undergo antigenic variation due to mutations or recombination events. This may result in different virulence factors or tropism for different host cells or tissues. For example, BToV strains differ in their ability to infect enterocytes or M cells of Peyer`s patches; HToV strains differ in their ability to bind to histo-blood group antigens as receptors; PToV strains differ in their ability to induce apoptosis or necrosis in infected cells.
• Co-infection with other pathogens: Toroviruses can interact with other enteric pathogens and modulate their pathogenesis. This may result in synergistic or antagonistic effects on viral replication or host immune response. For example, BToV can enhance rotavirus infection by increasing viral shedding or reducing interferon production; HToV can inhibit norovirus infection by competing for receptors or inducing interferon production; PToV can suppress transmissible gastroenteritis virus infection by inducing apoptosis or reducing viral shedding.
• Environmental factors: Toroviruses can be influenced by environmental factors such as temperature, pH

Toroviruses can cause gastroenteritis in both animals and humans, leading to diarrhea, dehydration, fever, and other symptoms. The severity and duration of the illness may vary depending on the host species, age, immune status, and viral strain.

In calves

Toroviruses cause diarrhea in calves by infecting villous and crypt enterocytes of the mid-jejunum, ileum, colon and cecum and inducing villous atrophy and necrosis of the crypts. Clinical signs are pyrexia, diarrhoea, dehydration, lethargy and depression in calves as well adults. In calves, it may lead to anorexia, mucoid faeces and neurological signs like generalised weakness, paralysis, inability to stand along with trembling and sudden death.

In humans

The clinical forms caused by human torovirus include acute diarrhea, persistent (chronic) diarrhea, necrotizing enterocolitis in children. Acute diarrhea is usually self-limiting and mild, lasting for 2 to 5 days. Persistent diarrhea may last for more than 14 days and is associated with malnutrition and immunodeficiency. Necrotizing enterocolitis is a serious condition that affects premature infants and causes inflammation and necrosis of the intestinal wall. Human torovirus infection may also cause respiratory symptoms such as cough, rhinorrhea, and wheezing.

Toroviruses can be detected in fecal samples or intestinal tissues of infected animals or humans by various methods. Some of the commonly used techniques are:

• Electron microscopy (EM): This method can directly visualize the virus particles with their characteristic torus-shaped nucleocapsids in the samples. However, EM requires specialized equipment and expertise, and may not be very sensitive or specific for torovirus identification.
• Immunofluorescence assay (IFA): This method can detect the viral antigens in the epithelial cells of the small intestine using fluorescent antibodies. IFA is more sensitive and specific than EM, but it also requires fresh or frozen tissue samples and skilled personnel.
• Enzyme-linked immunosorbent assay (ELISA): This method can detect the viral antigens or antibodies in the samples using enzyme-labeled antibodies. ELISA is a simple, rapid and sensitive technique that can be performed on various types of samples, such as feces, serum or saliva. However, ELISA may cross-react with other coronaviruses or toroviruses, and may need confirmation by other methods.
• Hemagglutination inhibition (HI): This method can detect the viral antibodies in the serum samples by measuring their ability to inhibit the hemagglutination of red blood cells by purified toroviruses. HI is a specific and quantitative technique that can also measure the antibody titers and seroconversion. However, HI requires purified virus antigens and standardized red blood cells, and may not be very sensitive for low antibody levels.
• Reverse transcription-polymerase chain reaction (RT-PCR): This method can detect the viral RNA in the samples by amplifying specific regions of the genome using primers and enzymes. RT-PCR is a highly sensitive and specific technique that can also differentiate between different torovirus strains or genotypes. However, RT-PCR requires high-quality RNA samples and specialized equipment and reagents, and may be prone to contamination or false positives.

Depending on the availability of resources and the purpose of diagnosis, one or more of these methods can be used to confirm torovirus infection in calves or humans. A combination of antigen detection and antibody detection methods can provide a more accurate and comprehensive diagnosis than a single method.

There is no specific antiviral treatment for torovirus infection in humans or animals. The main goal of treatment is to prevent dehydration and electrolyte imbalance caused by diarrhea and vomiting.

Some of the general measures that can be taken to treat torovirus infection are:

• Drink plenty of fluids, such as water, broth, or oral rehydration solutions (ORS) that contain electrolytes.
• Avoid foods or drinks that can worsen diarrhea, such as sugary, fatty, or spicy foods, dairy products, caffeine, or alcohol.
• Eat small and frequent meals that are easy to digest, such as rice, bananas, toast, or applesauce.
• Rest and avoid strenuous activities until the symptoms subside.
• Monitor the signs and symptoms of dehydration, such as dry mouth, thirst, reduced urine output, sunken eyes, or skin tenting.
• Seek medical attention if the symptoms are severe, persistent, or accompanied by blood in stool, high fever, severe abdominal pain, or signs of dehydration.

In addition to these measures, animals suffering from torovirus infection may benefit from:

• Fluid therapy to replace the fluid and electrolyte losses. This may be given orally or intravenously depending on the severity of dehydration.
• Antibiotics to prevent or treat secondary bacterial infections that may complicate the course of torovirus infection.
• Anti-inflammatory drugs to reduce the inflammation of the intestinal mucosa and alleviate the symptoms.
• Vaccination to prevent future outbreaks of torovirus infection in susceptible animals.

Torovirus infection is usually self-limiting and resolves within a few days to a week. However, it can cause serious complications and even death in some cases, especially in young, old, or immunocompromised individuals or animals. Therefore, it is important to seek timely diagnosis and treatment and follow preventive measures to avoid exposure to the virus.

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