VIROLOGY

L4 Picornaviradae, Caliciviridae and Coronaviridae

Picornaviridae

Feature of Picornaviruses

• Members of the family Picornaviridae are non-enveloped, positive sense single-stranded RNA (+ssRNA) viruses with an ICOSAHEDRAL capsid.

• Replication occurs in the cytoplasm in membrane-associated complexes, and infection is usually cytolytic.

• As they are positive sense RNA viruses, they go straight into protein synthesis once in the cytoplasm; this enables a relatively speedy replication cycle.

• They are prone to mutations (as are other RNA viruses), and this contributes to strain variation and immunodiversity.

• They are stable in the environment.

  • The stability of picornaviruses to environmental conditions is important in the epidemiology of the diseases they cause and the methods of disinfection.

  • Variations in their pH stability mean that only certain disinfectants are suitable for use against each virus, e.g., sodium carbonate (washing soda) is effective against foot-and-mouth disease virus but not effective against the swine vesicular disease virus.

• If protected by organic material such as mucus or feces and shielded from sunlight, picornaviruses are relatively heat stable.

• Consist of 12 genera, divided primarily according to difference in their genomes.

Pathogenesis of Picornavirus Disease

• Most picornaviruses are cytolytic.

• The viral RNA is released into the cell, probably through a membrane channel.

• Viral replication occurs in the cytoplasm.

• Release of the virus from the cytoplasm usually occurs when the cell lyses

• Localized infection is followed by viremia, and specific picornaviruses and picornavirus strains and subtypes can have specific tissue tropism, which reflect the virus’s disease manifestation in the host.

• With the exception of FMDV, picornaviruses typically infect a single or limited number of host species.

• Picornaviruses tend to be very contagious with transmission typically occurring through direct contact with infectious excretions or ruptured vesicles.

• Abrasions to the skin and mucosal surface can aid entry into the host.

• Given the non-enveloped nature of the virus, transmission through indirect routes from contaminated environments or fomites is also highly possible.

• Biting arthropods can facilitate mechanical transmission of the virus between animals.

• Aerosols, ingestions, and insemination/sexual are viable means of transmissions for picornoviruses.

• Some picornaviruses, notably FMDV, can produce persistent infections.

Significant Picornaviruses of Animals

Foot and Mouth Disease Virus (FMDV)

• FMDV is still a major global animal health problem.

• It is an exotic disease to Australia and New Zealand, but it is one of the most important exotic diseases in terms of potential damage to our economy.

• In general, FMDV infects a wide variety of cloven-foot domestic and wild animals including cattle, sheep, goats, pigs, and camels (BUT not horses).

• Mortality is low, but morbidity is high.

  • This means that virus shedding from affected animals is protracted and contributes to the spread of virus.

• Virus can persist in cats, dogs, rodents, and other small animals.

Features of FMDV

• There are seven serotypes of FMDV: O, A, C, SAT 1, SAT 2, SAT 3, + Asia 1.

  • These serotypes have differing geographic distributions. A large number of subtypes is recognized within each serotype. The tendency to mutate as evidenced by the number of serotypes creates difficulties in controlling this virus by vaccination.

• FMDV can survive extended periods in animal secretions and products.

  • Virus may be recovered from tears, nasal discharge, saliva, urine, feces, milk, vaginal discharge, semen, and membranes of aborted fetuses.

• It is sensitive to acid and alkaline conditions outside the range pH 6-9.

• Virus can remain infective on soil for 3 days in summer and up to 28 days in the winter.

• The survival of the virus in excretions depends upon temperature, pH, and humidity.

• It is inactivated by heating above 50°C.

• Epitheliotropic

Transmission of FMDV

• Low mortality and high morbidity mean infected animals become viral factories.

  • Therefore, viral shedding is protracted and contributes significantly to the spread of infection in an animal population.

• The virus is transmitted by:

  1. Direct contact

  2. Aerosol

  3. Fomites in contaminated environment

  4. Possibly arthropod

• The virus is considered highly contagious, with transmission between animals typically occurring through the inhalation of infectious droplets generated from ruptured vesicles around the mouth and muzzle.

• The virus can also be transmitted through the ingestion of contaminated food or fomites on contaminated equipment.

• FMDV can be transported over long distances via infected animals or their products, humans, vehicles, airborne spread (wind direction and speed are important issues), and possibly insects and birds.

Pathogenesis of FMDV

• FMDV usually enters via the respiratory tract with initial replication in the oropharynx.

• Viremia then occurs and this PRECEDES clinical signs by several days (2-8 days).

  • The virus may be isolated from the vagina, rectum, pharynx, blood, and milk during this time.

• Prior to, during, and after clinical signs subside, the tissue and secretions/excretions from the animal contain large amounts of virus are a source of virus to other animals.

• The FMDV virus may persist in the pharynx of recovered cattle for periods of up to 2 years. In sheep, they can carry the virus for up to 9 months. Pigs do NOT, however, become carriers.

• Virus rapidly moves from the blood during viremia to infect the epithelium of the oral cavity and feet.

• The characteristic epithelial lesions of FMD are the vesicle (i.e., vasicular), which forms as a consequence of both intracellular and intercullar edema.

• These vesicles rapidly rupture to leave erosions and ulcers in their affected epithelium.

• In young animals, FMDV can infect the heart musculature, causing degeneration and necrosis.

• Secondary bacterial infections can exacerbate and prolong the disease.

Diagnosis of FMD

• This is a notifiable disease!

• Australian Animal Health Laboratory must be called immediately. They will collect samples and conduct tests.

• Samples to test include vesicular fluid, epithelium tissue from ruptured vesicles, blood, fluids, and post-mortem samples.

• An ELISA test is available for use with vesicular fluid for viral antigen.

• Confirmatory diagnosis is based on the isolation of FMDV from samples of tissue or vesicular fluid. Epithelium collected from an unruptured or recently ruptured vesicle is ideal for laboratory processing.

• The preferred test for virus detection is RT-PCR.

• MUST be able to diagnose and distinguish FMDV from other vesicular diseases.

Control and Prevention of FMDV

• Control of FMD is difficult due to its highly contagious nature, multiple hosts, viral stability, multiple antigenic types, and short-term immunity.

• Vaccines have been produced and are used in affected countries.

  • These are usually inactivated vaccines of tissue culture origin, administered in adjuvant.

  • Vaccine must be of the appropriate serotype for that area. The safety of live attenuated vaccines is questionable, so their use is not widespread.

• In countries that are free from FMD, it is notifiable.

  • Affected and in-contact animals are slaughtered.

  • Following an outbreak, movement restrictions are applied, and infected premises must be thoroughly cleaned and disinfected.

  • Reserves of inactivated virus are maintained in several countries to provide an adequate supply of vaccine at short notice in the event of a major outbreak of the disease.

• Although ring vaccination around an affected premise may help limit the spread of the disease, it may also allow the development of the carrier state in animals subsequently exposed to the virus.

• The aim in carcass disposal is to minimize the possibility of further spread of disease from the carcasses.

  • Therefore, incineration (i.e., burning) is the best mode of carcass disposal

• Immunity is serotype specific, with protection against antigenically-similar subtypes within a serotype producing satisfactory protection lasts for up to 6 months.

• Foot and mouth disease virus has an ideal modus operandi for continued survival due to its environmental resistance, the ease of viral transmission, its rapid replication in many animal species at peripheral sites and its high morbidity.

Caliciviridae

Feature of Caliciviruses

• Members of the family Caliciviridae are small (25-40 nm in diameter), non-enveloped, positive sense single-strained RNA (+ssRNA) viruses with an ICOSAHEDRAL capsid, with a cup-shaped depression on the surface of the virion.

• Replication occurs in the cytoplasm, and virions are released by cell lyses (i.e., infection is cytolytic).

• Fairly resistant to heat + detergents and moderately pH stable (inactivated at pH 3, so don’t like acidic conditions)

• They are prone to mutations (as are other RNA viruses), contributing to strain variation.

• They are stable in the environment.

• Have a wide range of tissue tropism (i.e., tissue affinity)

Pathogenesis of Calicivirus Disease

• Caliciviruses replicate rapidly in the cytoplasm and are rapidly cytopathic.

• They are similar to picornaviruses in that they shut down host protein synthesis.

• Caliciviruses have a wide range of tissue tropisms (ie tissue affinity).

• Calicivirus can cause blistering of the skin and mucous membranes (e.g., mouth and appendages, FCV, VES), pneumonia (FCV), hepatitis (RHDV), or enteritis (CCV).

• The infidelity of their RNA polymerase enzymes results in many mutants being formed and the development of multiple strains. This can make vaccination against multiple strains difficult.

• Transmission is facilitated by ingestion or inhalation of infectious material from various types of infectious animal secretions or tissue.

• Due to the virus’s stability in the environment, indirect transmission through ingestion or aerosols generated from contaminated environments and fomites is also highly possible.

Significant Caliciviruses of Animals

Vesicular Exanthema of Swine Virus (VESV)

• VES is an acute viral disease characterized by the formation of vesicles on the mouth, lips, tongue, oral cavity, interdigital spaces and coronary band of the foot.

• First recognized in California in 1932 and again in 1952. By 1956, it was eradicated from USA.

• The virus appeared to spread from California to 48 other states of the USA by the ingestion of contaminated pork in garbage that was fed to pigs. It is suspected this occurred by throwing garbage from railway cars in the USA. The virus then spreads from pig to pig.

• This disease is possibly now extinct, but it remains on the list of differential diagnoses for FMDV.

• The natural disease appears to be specific for pigs.

• Clinically it is indistinguishable from foot-and-mouth disease, swine vesicular disease, and vesicular stomatitis.

• Incubation is 24-72 hours, and disease lasts 1-2 weeks. Infected swine have a rise in temperature lasting 6 days.

• The disease has low mortality and high morbidity (i.e., many affected but few die).

  • However, it causes serious weight loss in fat pigs, slow gains in feeder stocks, death in suckling pigs, and abortions in pregnant sows.

• The virus is suspected to have arisen from infected pinnipeds (seals and sea lions) that were washed up on beaches and then the carcasses fed to pigs.

• In 1972, the San Miguel sea lion virus (SMSV) was isolated from California sea lions, which had developed vesicles on their flippers. Premature parturition occurred in infected animals.

• Subsequently, SMSV has been isolated from a number of other marine mammals and from the opal eye fish.

• Strains of SMSV produce VES when inoculated into pigs, and it is thought that the original outbreak of VES arose through the feeding of uncooked swill containing meat from infected marine mammals.

• Vesicular fluid and the overlying flap of epithelium are rich in virus.

• Isolates can be determined by RT-PCR, ELISA, immunoelectron microscopy, and virus isolation in pig kidney cell lines.

• When examining EM of infected cell lines the virion appear as cisternae or crystalline arrays in the cytoplasm (i.e., sheets of virions)

Rabbit Hemorrhagic Disease Virus (RHDV)

• RHDV is an acute viral disease with very high mortality.

• It appears to be pathogenic for European rabbits only and was first identified in China in 1984. It is not known to infect other mammals.

• RHDV affects only rabbits over 2 months of age. Curiously, rabbits less than 2 months of age do not develop clinical disease following infection.

• In the peracute form of the disease, it causes a rapid onset of hemorrhage and necrosis in the lung and liver. Disseminated intravascular coagulation (DIC) with death occurs within 6-36 hours.

• The virus is shed in all excretions and secretion; however, transmission is mainly via the fecal-oral route. Infection may also occur by inhalation or through the conjunctiva.

• Mechanical transmission by arthropods has been demonstrated.

• The virus survives in the environment, and indirect transmission through contaminated foodstuff or fomites may occur.

• In subacute forms of the disease, rabbits have developed a serosanguinous nasal discharge and a variety of neurological signs. Mortality in infected rabbits approaches 100%.

• The virus was inadvertently released from a research facility in Australia during 1995 and subsequently illegally introduced into New Zealand in 1997 to decrease the wild rabbit population.

• In Australia, it has resulted in > 60% decline in rabbit numbers, and there was evidence of restoration of original habitats and Australian native species.

• Wild rabbit populations have now stabilized to 40-50% of the pre-released numbers in 1995 (~100-150 million).

• There is evidence of rabbits with resistance to the virus and circulation of benign/attenuated strains, all of which is reducing the viruses’ capacity to suppress wild rabbit population numbers.

History of the European Rabbit in Australia

• Thomas Austin, a wealthy Victorian grazier, introduced wild English rabbits into Australia in 1859 for sporting hunters.

• With no natural predators and litters of five or more baby bunnies seven times a year, there was a rabbit plague.

• By 1865, 20,000 rabbits were shot on his property. Farmers ripped their warrens, laid poison, and shot them, but still they multiplied.

• In the 1950's CSIRO introduced myxoma virus which killed millions of rabbits (myxomatosis).

• The idea of using myxoma virus to control rabbits was first raised in 1919 by a Brazilian Scientist H. B. Aragao, but was rejected at first by the Australian Government.

• Fourteen years later, a prominent pediatrician in Melbourne, Dame Jean Macnamara, persuaded the Government to undertake preliminary tests on its safety and trials were undertaken by CSIRO.

• The tests and conventional controls of trapping, shooting, and poisoning rabbits were disrupted by World War II. Rabbits flourished, causing widespread environmental destruction.

• After the war myxoma virus was evaluated in the field. Many of the early trials had been carried out in remote, arid areas, but in 1950 trials began in the Murray Valley.

• In early 1951 myxoma virus spread explosively through the Murray Valley, with mosquitoes as the major vector, and proved to be highly effective.

• Within 2 years of its spread, Australia's wool and meat production had recovered from the rabbit onslaught to the tune of $$68 million.

• Myxomatosis helped control rabbits for the following 50 years, although its initial effectiveness has waned.

• By 1995 rabbits had multiplied to an estimated 300 million.

• CSIRO scientists were asked to solve this problem.

• In March 1995, a quarantine station was set up on tiny Wardang Island off the coast of South Australia to test Rabbit calicivirus, which had kept down rabbit populations in Europe.

• It was due for release in 1998, but, after only 6 months, it escaped from the island, most likely carried by insects.

• CSIRO researchers have since added calicivirus to augment control of the rabbit and are now exploring new biotech methods of control specifically to prevent them from breeding.

• As the rabbits disappeared, the barren landscape flourished once again.

• There was some criticism about a reduction in food for foxes and eagles that could result in them turning to native fauna for food.

• Up till now the calicivirus has been most successful in dry regions.

• Scientists are also aware that because myxomatosis was only effective for 15 to 20 years, resistance to calicivirus is known being seen in rabbits.

• However new strains of RHDV have emerged some of which infect kitten rabbits as young as 3 weeks of age. This new strain (RHDV2) has been able to have a sustained impact on wild European rabbit populations in Australia.

Control and Prevention of RHDV

• The introduction of the virus has required that pet, commercial and research rabbits be vaccinated against RHDV.

• A killed vaccine (Cylap®) is available that protects from the disease and is routinely used in practice. Rabbit at about 10 weeks of age are vaccinated with annual boosters recommended.

• A new vaccine that can protect against both RHDV1 and 2 (Filavac K C+V) is currently under assessment by the APVMA with a potential release for use in pet rabbit in Australia in 2024.

• Control can also be achieved through strict quarantine and isolation to prevent transportation of RHDV-contaminated materials into commercial rabbitries and pet rabbit in rural settings.

• Ectoparasite control can also be useful in protecting pet rabbit in rural settings becoming infected through mechanical transmission of the virus via arthropods from wild European rabbits.

Coronaviruses

• Feline Infectious Peritonitis Virus → enteritis in cats, peritonitis and immune mediated vasculitis, invariable fatal

• Zoonotics Coronovirus → SARS, MERS, COVID-19

L6 Flaviviridae, Togaviridae and Reoviridae Learning Outcomes

• By the end of this lecture, you should be able to:

  1. Recognise the unique features of flaviviruses, togaviruses and reoviruses.

  2. Recognise the significant animal pathogens within each viral group.

  3. Understand the pathogenesis and epidemiology of animal diseases caused by flaviviruses, togaviruses and reoviruses

  4. Comprehend the various strategies used to diagnose, control and prevent diseases caused by flaviviruses, togaviruses and reoviruses in animal populations.

Suggested readings:

• Veterinary Microbiology and Microbial Disease (2nd Edition), Quinn PJ, Markey BK, Leonard FC, Fitzpatrick ES, Fanning S, Hartigan PJ, Wiley-Blackwell, Iowa (2011):

  • Chapter 81: Flaviviridae

  • Chapter 82: Togaviridae

  • Chapter 69: Reoviridae

• Veterinary Microbiology (2nd edition), Hirsh DC, MacLachlan, Walker RL, Blackwell, Iowa (2004):

  • Chapter 58: Togaviridae and Flaviviridae

  • Chapter 63: Reoviridae

Viruses Covered in Lecture

• Japanese encephalitis virus → mosquito borne viral disease of humans and animals that occurs throughout much of Asia, causes abortion in pigs and encephalitis in humans and horses

• West Nile virus→ causes encephalitis in humans and horses, natural reservoir birds, transmitted by mosquitoes, ongoing problem in the U.S.A.

• Kunjin virus → causes neurological disease in horses in Australia, closely related to West Nile virus

• Bovine viral diarrhoea virus → diarrhoea and mucosal disease, congenital infections may result in abortion

• Western, Eastern and Venezuelan equine encephalitis viruses (WEE, EEV, VEE) → Mosquito borne viral pathogens (arboviruses) of horses and man causing encephalitis and other neurological syndromes.

• Bluetongue virus → causes fever, congestion; oedema and haemorrhage in sheep, affect sheep salivate profusely develop cyanosis (bluetongue) and may well die of aspiration pneumonia

• Rotavirus→ causes enteritis and diarrhoea in all domestic animals especially those that are intensively reared

L6 Orthomyxoviridae, Paramyxoviridae and Rhabdoviridae Learning Outcomes

• By the end of this lecture, you should be able to:

  1. Recognise the unique features of orthomyxoviruses, paramyxoviruses and rhabdoviruses

  2. Recognise the significant animal pathogens within each viral group

  3. Understand the pathogenesis and epidemiology of animal diseases caused by orthomyxoviruses, paramyxoviruses and rhabdoviruses

  4. Comprehend the various strategies used to diagnose, control and prevent diseases caused by orthomyxoviruses, paramyxoviruses and rhabdoviruses in animal populations.

Suggested readings:

• Veterinary Microbiology and Microbial Disease (2nd Edition), Quinn PJ, Markey BK, Leonard FC, Fitzpatrick ES, Fanning S, Hartigan PJ, Wiley-Blackwell, Iowa (2011):

  • Chapter 71: Orthomyxoviridae

  • Chapter 72: Paramyxoviridae

  • Chapter 73: Rhabdoviridae

• Veterinary Microbiology(2nd edition), Hirsh DC, MacLachlan NJ, Walker RL, Blackwell, Iowa (2004):

  • Chapter 59: Orthomyxoviridae and Bunyaviridae

  • Chapter 60: Parammyxoviridae, Filoviridae and Bornaviridae

  • Chapter 61: Rhabdoviridae

Viruses Covered in this Lecture:

• Swine influenza virus → acute respiratory disease in pigs

• Equine influenza virus → upper respiratory disease in horse

• Avian influenza virus→ respiratory, enteric and neurological disease in domestic birds, some strains can cause disease in humans with high mortality

• Hendra virus→ causes a hyper acute fatal pneumonia in horses and can infect humans with a 50% mortality rate, viral reservoir is fruit bats.

• Nipah virus → causes fatal pneumonia and encephalitis in pigs and can infect humans with a high mortality rate, viral reservoir is fruit bats.

• Rabies virus→ causes a fatal neurological disease in all warm-blooded animals, wild carnivores (i.e. foxes) major reservoirs of infection

• Australian bat lyssavirus→ causes neurological syndromes and death in Australian bats; can be transmitted and can cause neurological disease in humans in close contact with sick bats

L7 Cell culture Learning Outcomes

• By the end of this lecture, you should be able to:

  1. Describe the principles behind viral replication in the laboratory.

  2. Recognise the various methods used to propagate viruses in the laboratory.

  3. Recognise the tools and biosafety measures needed to propagate viruses in a safe and sterile manner in the laboratory.

  4. Describe the virus-cell interaction that can be observed in the laboratory.

  5. Recognise the various methods used to detect viral growth in the laboratory.

Suggested readings:

• Veterinary Microbiology and Microbial Disease (2nd Edition), Quinn PJ, Markey BK, Leonard FC, Fitzpatrick ES, Fanning S, Hartigan PJ, Wiley-Blackwell, Iowa (2011):

  • Chapter 57: Propagation of viruses and virus-cell interaction

• Veterinary Microbiology(2nd edition), Hirsh DC, MacLachlan NJ, Walker RL, Blackwell, Iowa (2004):

  • Chapter 3: Laboratory diagnosis

Topics and Issues to be Covered in Lecture:

• Virus propagation

  • Tissue culture

    • Covering requirements and techniques used in tissue cultures, various media and laboratory containment facilities needed and how to avoid contamination

    • The various cell types that can be used in viral tissue culture and the differences and pros and cons of continuous cell lines and primary cell cultures.

  • Examine the use of embryonated eggs and experimental animals in viral propagation in the laboratory and explore the reasoning behind the continuation of these techniques.

    • Detection of viral growth

      • Defining and differentiating cytopathic and non-cytopathic viruses.

      • Examine the following methods used to detect viral growth in the laboratory; plaque assays, microscopy and cytopathic effect.

L8 Immunological and molecular techniques in virology

• By the end of this lecture, you should be able to:

  1. Describe the methods used to collect samples for virology diagnostics.

  2. Describe the methods used to isolate and visualise viruses in the laboratory.

  3. Recognise and understand the principles underpinning the various immunological methods used in virology diagnostics.

  4. Recognise and understand the principles underpinning the various nucleic acid molecular methods used in virology diagnostics.

Suggested reading:

• Veterinary Microbiology and Microbial Disease (2nd Edition), Quinn PJ, Markey BK, Leonard FC, Fitzpatrick ES, Fanning S, Hartigan PJ, Wiley-Blackwell, Iowa (2011):

  • Chapter 59: Laboratory diagnosis of viral infections

• Veterinary Microbiology(2nd edition), Hirsh DC, MacLachlan NJ, Walker RL, Blackwell, Iowa (2004):

  • Chapter 3: Laboratory diagnosis

Topics and issues to be Covered:

• Methods of sample collection and laboratory transport for virology diagnostics

• Methods used to isolate and visualise virus.

  • Cell culture, eggs and animals

  • Cytopathic effects on cells

  • Electron microscopy

• Immunological methods used to detect, identify and quantify viral infections and host responses.

  • Principles of antibody-antigen reaction in viral diagnostics.

  • Application of immunological diagnostics methods in virology.

    • Haemagglutination and haemagglutination inhibition assays

    • Serum neutralisation assays

    • Monoclonal and polyclonal antibodies

    • Enzyme Linked Immunosorbent Assay (ELISA) and Western Immunbot Assay.

    • Complement fixations test

    • Staining using fluorescence antibody

• Nucleic acid techniques used to detect, identify and quantify viral infection

  • Principles of nucleic acid interactions and amplification in viral diagnostic

  • Application of nucleic acid diagnostics methods in virology.

    • Nucleic acid probes

    • Polymerase chain reaction (PCR)

    • Reverse Transcriptase (RT) -PCR

    • Real time or quantitative (Q) -PCR

    • Sequencing

L9 Parvoviridae and Papillomaviridae

• By the end of this lecture, you should be able to:

  1. Recognise the unique features of parvoviruses and papillomaviruses.

  2. Recognise the significant animal pathogens within each viral group.
     3.