Coronavirus

Question 1

What is central to the Baltimore Classification diagram?

Answer 1

How the virus is going to generate proteins from mRNA.

Question 2

What order of virus does coronavirus belong to?

Answer 2

Nidovirales.

Question 3

1. What are nidovirales?
2. Which group of Baltimore Classification system is coronavirus in?

Answer 3

1. Form a phylogenetically compact but diverse group of enveloped, positive-stranded, RNA viruses.
   Nido: from Latin Nidus = ‘nest’ and refers to the synthesis of a 3’-coterminal, nested set of mRNAs, hallmark of nidovirus transcription.
2. 4.

Question 4

1. Betacoronaviruses.
2. Gamma coronaviruses.
3. Alphacoronaviruses.

Answer 4

1. Mouse Hepatitis Virus, SARS-CoV, SARS-CoV-2, Human CoV OC43, BCoV.
2. Infectious bronchitis virus (IBV).
3. Transmissible Gastroenteritis Virus (TGEV) (pigs), Porcine Respiratory CoV, Canine and Feline CoV, Human CoV (229E, NL63).

Question 5

What do coronaviruses look like?

Answer 5

Corona = “halo” refers to distinctive appearance of surface projections that create an image reminiscent of the solar corona.
Structure = enveloped virus with characteristic large peplomers (Spike-protein) and a core containing +RNA helical nucleocapsid.
80-120nm diameter.

Question 6

Nidovirales morphology.

Answer 6

Coronavirus (as discussed).
Torovirus – crescent shaped.
Bafinivirus and ronivirus – rod shaped.
Arterivirus – small circular shaped.

Question 7

1. Coronavirus spike structure.
2. What do coronaviruses use these spikes for?

Answer 7

1. Trimer.
   3 receptor binding S1 domains.
   S2 domains anchor spike into viral membrane.
2. To attach to cell entry receptors on the host cells (15-20 contact points), and be taken up by endocytosis.

Question 8

Coronavirus replication.

Answer 8

• Spike protein on the virion binds to ACE2 (cell surface protein) and TMPRSS2 (enzyme) helps virion enter.
• Virion releases its RNA into host cell.
• Some RNA translated into proteins by the cell’s machinery, incl. production of
• Some of these proteins form a replication complex to make more RNA.
• Proteins and RNA assembled into new virion in Golgi.
• New virion released.

Question 9

1. What are syncytia?
2. How does coronavirus form syncytia?

Answer 9

1. Multinucleated cells.
2. Coronavirus infects host cell. Infected host cell produces viral proteins. Some of these are Spike glycoproteins. Spike glycoprotein expressed on surface of infected host cell. Infected cell could be sitting next to another cell, which is likely to have an ACE2 receptor on its surface. The contact between Spike and ACE2 causes infected cell and normal host cell to fuse together. This forms giant multinucleated cells (syncytia) to be formed.

Question 10

What is the benefit of virus budding from an intracellular site?

Answer 10

Virus does not have to kill the cell and can avoid exposing its peplomers on the cell surface where antibodies may bind and mediate killing of the virus.

Question 11

1. How many genes does coronavirus genome code for?
2. What do genes at the 5’ end code for?
3. What do genes at the 3’ end code for?
4. Cap and tail?
5. Open reading frames?

Answer 11

1. 6-7.
2. Non-structural proteins e.g. enzymes.
3. Structural proteins.
4. 5’ cap and poly A tail.
5. Expressed in 3’ end from a nested set of sub-genomic RNAs.

Question 12

1. What is a 5’ cap?
2. What is poly A tail?

Answer 12

1. Protection of the growing RNA chain from degradation by nucleases. Aids stability.
2. Protects the RNA and increases stability and plays a role in the initiation of translation.

Question 13

1. What protein is made shortly after virus infects host cell? – purpose?
2. Genome replication.

Answer 13

1. RNA dependent RNA polymerase (RdRp). – to start replicating its genome.
2. • The + strand is first copied into a full length negative strand.
     - The -ve strand is then copied into a full length + strand.

Question 14

1. What are the nested transcripts of nidoviruses also referred to as?
2. How are these created?

Answer 14

1. Sub-genomic RNAs.
2. Discontinuous transcription.
   During transcription of negative strand RNA, polymerase have chances to pause on transcription-regulating sequences (TRS) and jump to leader TRS, thereby creating a major deletion.

Question 15

What is a transcription-regulating sequence (TRS)?

Answer 15

An area of the genome where there is a lot of structure (RNA folding) so difficult for RdRp to get past it. If cannot get past, RdRp dissociates.

Question 16

Porcine Transmissible GastroEnteritis Virus…
1. How does infection occur?
2. How does the virus enter the cells?
3. Incubation period?
4. Symptoms?
5. Most at risk / susceptible?
6. Adults?

Answer 16

1. By ingestion (faecal-oral).
   - Virus is acid labile and is protected by milk and food as it passes through the stomach.
2. Via attachment to receptor Aminopeptidase-N (APN) on epithelial cells (enterocytes) on intestinal villi.
3. 18-72hrs.
4. Vomiting, profuse watery diarrhoea, rapid weight loss and dehydration.
5. Neonates.
6. Asymptomatic.

Question 17

1. Effect of TGEV on intestines?
2. Subsequent effect on the host?

Answer 17

1. Destroys integrity of intestinal architecture and disrupts intestinal homeostasis by killing to enterocytes of the villi (villous atrophy).
2. • Loss of fluid absorption.
     - Undigested lactose with secondary bacterial fermentation in the colon.
     - Osmotic effects compound the diarrhoea – typical yellow milky intestinal contents.
     - Recovery rapid (providing that death due to dehydration has not occurred).

Question 18

How can TGEV be diagnosed?

Answer 18

• Histological staining of intestines from neonatal piglets that have died due to infection. – using immunofluorescence or immunoperoxidase.
• Antigen capture ELISA to detect virus in faeces.
• Isolate virus from dead pigs, usually from kidneys, thyroid or testicle cells samples (because virus can grow in these tissues).
• RT-PCR can be used to detect the virus.

Question 19

How can we protect against TGEV?

Answer 19

• Secretary (actively from mucosa) IgA antibody is protective, systemic IgG is not.
• Vac of sows w/ live attenuated TGEV to boost maternal immunity can be used.
• Control of TGE by depopulation and disinfection of affected pens.
• Removal of Seropositive adults. Virus can be secreted for >100days in the carrier.

Question 20

1. What are the 2 biotypes of feline coronavirus (FCoV).

Answer 20

1. Feline Infectious Peritonitis Virus (FIPV).
   Feline Enteric Coronavirus (FECV).
   FIPV = mutation of FECV.

Question 21

1. What does FIP result in?

Answer 21

1. often systemic and fatal infection.

Question 22

1. How is feline coronavirus transmitted?

Answer 22

1. Faecal-oral. Typically shed in faeces by healthy cats and transmitted by the faecal-oral route to other cats.
   Transmission rate is much higher in environments with multiple cats compared to single-cat environments.

Question 23

1. Where does FECV infection occur?
2. Signs?
3. How can FECV develop?

Answer 23

1. Gastrointestinal epithelial cells.
2. Few outward signs, usually chronic. Virus excreted in faeces of healthy carrier.
   May occasionally experience brief bouts of D+ and/or mild URT signs from which they recover spontaneously.
3. Can develop into FIP, which is progressive and almost always fatal.

Question 24

1. What 5 cats infected w/ FECV develop FIPV?
2. How does FECV develop into FIPV?

Answer 24

1. 10%
2. One or more mutations of the virus can alter its biological behaviour, resulting in WBCs (e.g. macrophages) becoming infected, which helps the virus throughout cat’s body. An intense inflammatory reaction to FIPV occurs around vessels in the tissues where these infected cells locate, often in the abdomen, kidney or brain.

Question 25

Why is the infection on macrophages so intense?

Answer 25

FIPV infects macrophages more easily in presence of antibody as antibodies raised against spike protein can enhance infection -- Antibody Dependent Enhancement (ADE). Antibodies produced will bind to the virus and they will enable the virus (via different receptor) to latch onto a macrophage, which will take up the antibody-virus complex, where the virus is then allowed to replicate in the macrophage.

Question 26

FCoV diagnosis.

Answer 26

- Histological examination can confirm presence of coronavirus infection of macrophages within lesions. - ELISA does not distinguish between FECV and FIPV. - Isolation of virus from nasal, oropharyngeal, and ocular swabs; lung and trachea from necropsied cats. -- Virus can be propagated in cell cultures where viral cytopathic effect (plaque assay) is seen. Antiserum against FIPV can be used to neutralise virus.

Question 27

Protection against FCoV?

Answer 27

Vac -- commercially available in USA, Canada and Europe. -- Contains temp sensitive modified live mutant strain of coronavirus for intranasal administration. -- Not licensed for kittens younger than 16 weeks old because protection not been demonstrated in these young animals. -- Developed to avoid induction of high serum antibody levels as these may promote ADE of infectivity. -- Not very good -- Variable success.