Viral Disease

Question 1

List the 5 categories of testing techniques available for the diagnosis of viral disease

Answer 1

1. Virus isolation in cell culture
2. Electron microscopy
3. Specific viral antigen detection
   
   • ELISA
   • Fluorescent antibody
   • Immunoperoxidase
   • Immunohistochemistry
4. Nucleic acid detection
   
   • PCR
   • In-situ hybridisation
5. Serological testing for specific antibodies

Question 2

What is meant by recombination in virology.

How may recombination occur naturally, providing an example

Answer 2

• Recombination is a process by which pieces of DNA or RNA are broken and recombined to form new alleles
• The process is different from mutation in that it “shuffles” the DNA / RNA to produce a new allele
• This process can occur naturally when two separate strains invade the host cell at the same time
• The formation of FCoV class II were likely a recombination of FCoV I and CCoV. This recombination process changed the spike protein gene in the FCoV class II versus the class I virus.

Question 3

Briefly explain the use of serological testing to assess vaccine protection

Answer 3

• Serological testing utilises purifed antigens of a specific virus or disease to “capture” antibodies in the serum of the patient tested
• Serological tests can identify the presence of different antibody types such as IgM, IgG, IgA
• Specific concentrations (titres) of the relevant antibody can be determined
  
  • The presence of a specific defined titre level can be considered to be protective against the disease caused by the specific antigen (virus or other)
• Other factors need to be considered over and above the specific antibody titre
  
  • breed
  • environment (potenital risk of exposure)
  • immune status including potential immunosuppression

Question 4

Briefly describe the process of PCR and what is tested

Answer 4

• Polymerise chain reaction (PCR) testing utilises heat cycling to allow rapid multiplication of small segments of DNA
  
  • The small segment of DNA is usually a small specific segment of an organism or product produced by an organism such as a toxin
• A DNA template is used to identify the segments of DNA to amplify
  
  • This template can detect even minute amounts of the appropriate DNA
• The DNA is initially denatured by using a high temperature (~90 degrees)
  
  • This breaks the hydrogen bonds and yields two separate DNA strands
• The sample is cooled to 50-65 degrees to allow binding of primers to the 3’ end (annealing)
• The sample is cooled further to allow the specific DNA polymerase to synthesise a new DNA strand, complimentary to the original
• repeated cycles leads to exponential amplification as follows
  
  • single piece of DNA = 2 strands
  • 2 to the power of the number of cycles
  • 2³⁰ = 1,073,741,824 copies

Question 5

How does RT-PCR differ from traditional PCR

Note specific sample handing differences

Answer 5

• RT-PCR is used to identify specific RNA sequences
• The process is essentially the same as for PCR following the initial step
• The RNA segment of interest is initially converted into a DNA segment via the use of the enzyme reverse transcriptase

RNA is more fragile than DNA, so denaturing or degradation of the sample needs to be avoided. This can lead to an increased false negative rate

Question 6

Describe the retrovirus replication cycle

Answer 6

• Virus particle binds to a CD4⁺ surface molecule
  
  • This allows for binding and fusion of the virus envelope with the cell membrane
• RNA genetic material enters the cell
• Reverse transcriptase converts the viral RNA to DNA
• The DNA translocates into the host cell nucleus
  
  • With some viruses this can only occur during mitosis while in other such as HIV, penetration of the nuclear membrane is independent of cell cycle
• The viral DNA is integrated with the host nuclear DNA
• The viral DNA undergoes transcription to RNA which then translocates back into the cytoplasm
• Splicing of the transcripts occurs
• Translation of the RNA enables formation of new viral proteins and membranes
• The new viral particles are assembled and released via budding of the host cell membrane

Question 7

Based on the known mechanisms of infection and replication of viruses, list some of the potential targets for anti-viral medications

Answer 7

• Nucleoside reverse transcriptase inhibition
• Non-nucleoside reverse transcriptase inhibition
• Nucleoside analog DNA/RNA synthesis inhibition
• Nucleotide synthesis inhibitors
• Receptor antagonists
• Neuraminidase Inhibitors
• Ion Channel Blockers
• Peptides
• Immune modulation - interferons
• Cytokines / growth factors
• Inducers of cytokines / interferons

Question 8

Describe the mechanism of action of and clinical indications for the use of famcyclovir

Question 9

Describe the mechanism of action of and clinical indications for the use of remdesivir

Answer 9

• Remdesivir is a nucleoside analogue prodrug
• The prodrug is cleaved to form GS-441524 monophosphate
• This compound is delivered into the cell where is is converted to GS-441524 triphosphate
  
  • This compound is a ribonucleotide analogue inhibitor of viral RNA polymerase
• Inhibition of RNA polymerase, inhibits the replication cycle of the target virus
• A study from Pedersen et al in 2019 JFMS looked at a 12 week course of GS-441524 in 26 cats, 25 of which were long term survivors

Question 10

Describe the mechanism of action of and clinical indications for the use of oseltamivir (Tamiflu)

Answer 10

• Oseltamivir is a neuraminidase inhibitor
• Active against the viruses that contain neuraminidase - influenza, parainfluenza and paramyxoviruses
• Competitive inhibitor of sialic acid, part of the viral enzyme that cleave sialic acid residues on the cell surface
• This blocks the viral progeny from being able to leave the host cell
• May be useful in treatment of avian influenza in cats
• Might also be effective for canine influenza, but this is generally a mild and self limiting disease

Question 11

Describe the mechanism of action of and clinical indications for the use of feline interferon omega

Answer 11

• Feline interferon omega is a recombinant product produced by baculoviruses that contain the feline IFN product that replicate in silkworms after infection
  
  • Feline IFN is then purified out of the homogenised silkworm preparation
• IFN-o has antiviral and anti-tumour activities in vitro
• Distirbuted primarily into the liver and kidneys
• Catabolized rapidly, mainly in the kidneys
• Excreted by the kidneys with minimal residual accumulation
• Not shown to be beneficial in the treatment of FIV when used in an acute course
• Shown to provide a survival benefit in FeLV, but mechanism was not proven
• Not beneficial for the treatment of FHV associated conjunctivitis
• Placebo controlled double blinded study on FCoV showed no survival benefit with the addition of Feline IFN compared to placebo
• Survival benefit shown in a study that assessed acute CPV infection in dogs when compared to placebo
  
  • While no controlled studies

Question 12

Describe the mechanism of action of and clinical indications for the use of L-Lysine

Answer 12

• Reduced viral replication is attributable to antagonism of arginine by excess lysine
• ~28% of the human host cell protein fraction of the histone layer around DNA is lysine, 3-4% arginine
  
  • These ratios are reversed for the capsid coat around the DNA core of the herpervirus
• Lysine may act as an analog for arginineor compete for cellular transport mechanisms
  
  • Reduced production of the capsid coat and reduced viral replication
• Aopted from human medicine where is suppreses clinical manifestation of herpesvirus infection.
• Studies have shown both delayed signs of infection and reduced severity of signs in both acute disease and after reactivation of latent infection
• Can also reduce viral shedding

Question 13

Briefly describe the canine coronavirus structure and etiology

Answer 13

• Several strains have been identified - CCoV I and CCoV II with two strains of CCoV II noted as a and b
  
  • These canine coronaviruses are closely related to the feline CoV and a transmissable GE virus of swine
• Single stranded, positive sense RNA, enveloped with large numbers of spike proteins on the outer capsid
  
  • Susceptible to detergents and inactivated in the environment after ~40 hours at room temperature, 60 hours when refrigerated and < 1 hour at temperatures > 65 degrees C
• Replication occurs within the host cell cytoplasm
  
  • RNA is directly transcribed by the ribosomes in the RER

Question 14

Describe the pathogenesis of Canine Enteric Coronavirus

Answer 14

• 1-3 day incubation with shedding from 3-14 days in the faeces
  
  • PCR has detected virus particles in the faeces for up to 6 months
• After ingestion, they are taken up by the mature intestinal epithelial cells
  
  • Also see uptake by the M cells of the Peyers’patches with virus antigen transported to the underlying lymphoid tissue
  • This may help lead to latency of infection
• The virus rapidly replicates in the mature epithelial cells while the crypts are spared
  
  • Epithelial cells rupture and the brush border microvilli become short, distorted or lost
    
    • This leads to markedly reduced absorptive capacity
• Crypt hyperplasia develops to replace the lost mature cells - see various degrees of villus atrophy and fusion
• Mononuclear infiltration of the LP occurs

Question 15

Briefly note the eitiology and pathogenesis of canine pantropic coronavirus

Answer 15

• Pantropic Coronavirus arised from a mutation from a type IIa canine coronavirus
• Alterations in the CCoV type IIa enable the pantropic strain to invade and infect tissues remote from the GIT
  
  • This ability is likely derived from a mutation int he spike protein gene
• The clinical disease is more similar to parvovirus than enteric coronovirus type I
• Severe haemorrhagic enteritis with serosanguinous abdominal effusion can be seen with pulmonary consolidation, renal infarction and abdominal lymphadenomegaly
• Viral antigen can be detected by IH staining within macrophages in inflammatory sites and within arterial walls
• The disease has many characteristics in common with FIP

Question 16

Briefly discuss the aetiology of feline parvovirus and the disease feline panleukopenia

Answer 16

• Non-encapsulated, single stranded DNA virus, genetically very similar to other parvoviruses including CPV
  
  • The feline disease can be caused by canine parvovirus
• FPV can survive up to 1 year at room temperature in organic material or on solid fomites
  
  • 5-10 months outside in organic material
  • Heat and drying accelerate inactivation
• Can survive ammonium disinfection, but killed by bleach at 5.25 %

Question 17

Discuss the epidemiology of feline parvovirus infection

Answer 17

• FPV is essentially ubiquitous int he environment and can infect all members of the family Felidae
• Virtually all susceptible cats are exposed within the first 12 months of life
  
  • 75% of unvaccinated, clinically healthy cats have demonstrable antibody titres by 1 year of age
• Disease outbreaks tend to parallel the seasonal nature of susceptible newborn kittens
• Short shedding time and prolonged environmental contamination mean that transmission is usually via contact with contaminated premesis
  
  • Fomites play an important role in transmission
  • Insects are also likely to play an important role in transmission in warmer months
• Virus particles are present / shed in all bodily secretions in the acute stage of disease, though the largest quantities are shed in the faeces
• In utero transmission occurs
  *

Question 18

Discuss the pathogenesis of feline parvoviral infection

Answer 18

• FPV requires host cell DNA polymerase to replicate, so targets rapidly dividing cells
  
  • These occur in the GIT crypts, bone marrow and lymphoid tissues
  • With late prenatal or early neonatal infeciotn, neurological targeting is also seen
• Oral exposure - replication in the oropharyngeal lymphoid tissue ⇒ plasma viraemia between 2-7 days disseminates the viral particles
  
  • Lymphoid tissue necrosis followed by proliferation
  • Thymid involution and degeneration (< 9 weeks of age)
  • Prenatal infection can result in immunosuppression and immune tolerance
    
    • This can lead to prolonged shedding
• The combination of enteric mucosal barrier loss and panleukopenia predisposes to secondary infection or endotoxemia with subsequent DIC
• Co-infections can increase the severity of disease
  
  • These can also damage intestinal epithelial cells, stimulating more rapid cell division

Question 19

Describe the different presentations and clinical signs with feline parvovirus

Answer 19

• Prenatal or early neonatal infection can cause neurological signs
  
  • Cerebellum is most often affected as it develops during the late prenatal and early neonatal phase (up to 9 days of age)
  • Cerebrum, optic nerve / retina, spinal cord - hydrocephalus and hydrancephaly can be seen with pre-natal infection
• In utero infection
  
  • Early fetal loss and resorption, abortion or birth of mummified foetus
• Enteric infection
  
  • Common - when infection acquired post-natally after ~ 10 days of age
  • Due to rapid replication of virus within the crypt cells
• Panleukopenia
  
  • Rapid replication in the bone marrow stem cells or early differentiated granulocytes, monocytes and lymphocytes

Question 20

Describe the routine laboratory findings in cats with clinical feline parvovirus infection

Answer 20

• The laboratory findings are dependent on the severity of infection together with the age at which infection occured
• For the classical post-natal / early infection that causes panleukopenia and enteritis
  
  • Neutropenia initially due to consumption
  • Leukopenia follows due to bone marrow damage
  • Neutrophilia follows if there is a recovery
  • Transient mild decrease in HCT and reticulocytes
  • Variable thrombocytopenia - from bone marrow injury or due to secondary coagulopathy / DIC
  • Biochemistry changes are non-specific - increases in liver enzymes, decreases in glucose reflect the clinical course of disease

Question 21

How can a diagnosis of feline parvovirus be confirmed

Answer 21

1. Faecal ELISA - identification of specific viral antigen
   
   • The canine parvovirus SNAP test has good cross sensitivity at the outset of the disease
   • Low viral load in the faeces following 24-48 hours post-inoculation (often prior to the onset of signs) means a diagnosis may be missed if tested later in the disease process
2. Serology
   
   • Rarely indicated for clinical cases
   • Single titres do not differentiate between clinical disease and post exposure
   • A four-fold rising titre 2 weeks apart can confirm active infection - though the kitten would have to survive!
3. Virus isolation
   
   • Not routinely used in clinical cases
4. Genetic detection
   
   • PCR - can detect specific parvovirus strain
   • Highly sensitive test that can detect virus shedding for significantly longer than ELISA
   • May detect subclinical shedding and can detect virus following MLV vaccination
   • A positive result needs to be interpreted together with the clinical signs (prevalence) to minimise the risk of a false or clinically irrelevant positive

Question 22

List and briefly describe the potential treatment options for kittens with clinically significant feline parvovirus infection

Answer 22

• Treatment is required for active infection causing enteritis and panleukopenia
• The majority of the treatment options are directed at supportive care
  
  • Subcutaneous or IV fluids
  • Antiemetics for vomiting
  • Gastroprotection (if not vomiting)
    
    • Bismuth compounds?
  • Plasma or whole blood transfusion
  • Heparin therapy if there is evidence of DIC and thrombocytopenia
  • Broad spectrum antibacterials
    
    • Third generation or extended penicillins with metronidazole or clindamycin with aminoglycoside
  • B-vitamin (combination) therapy
    
    • Due to anorexia and loss in diuresis / reduced absorption
  • IFN-omega
    
    • Studies in dogs have shown a protective effect
  • Nutrition
    
    • Especially after the initial period of instability and severe disease to assist recovery

Question 23

Brief notes on feline rotavirus epidemiology, clinical findings and therapy

Answer 23

• Rotaviruses have been isolated from many species and can be idenitifed by EM
• Rotaviruses are the main enteric pathogen in humans and livestock
• Feline rotavirus is essentially ubiquitous with up to 100% of the feline population being seropositive
• Enteric disease is generally mild to subclinical
• PCR, EM, ELISA or gel electrophoresis can be used to identify the RNA from the virus
  
  • Rarely utilised in the feline population
• The virus causes intestinal villi swelling and inflammation but not impairment to mucosal integrity
• Therapy is largely supportive and no vaccine is available

Question 24

Briefly describe the aetiology of feline coronavirus infection and FIP

Answer 24

• FeCoV is a large, encapsulated, positive sense single stranded RNA virus
• Coronavirus have the largest RNA structure of all known viruses containing the following:
  
  • Replicase polyprotein
  • Structural proteins: spike protein, M (matrix) protein, E (envelope) protein, N (nucleocapsid) protein,
  • Non-structural proteins
• FIP may arise from de novo mutation of wild type FCoV WITHIN the host cat, as opposed to direct transfer of FIP causative FCoV from cat to cat
  
  • This is questioned with a genetic study suggesting the FIP strains were more likely ancestrally derived
• A deletion mutation in the 3c non-structural protein may help the virus invade tissues other than the gut, while an intact 3c protein is necessary for gut replication
• These RNA viruses are particularly prone to mutation and multiple strains/variants can be seen within host and even within the same granuloma
• Different variants may have differing ability to replicate in monocytes / macrophages
• Viral load has an impact on development of FIP
• There are two serotypes - FCoV I and FCoV II (likely a recombinant of FCoV I and CCoV
  
  • Both serotypes can cause FIP

Question 25

Discuss the potential risk factors for cats to develop FIP

Answer 25

• Age: < 2 years of greatest risk
  
  • Second peak in cats > 10 years of age
• Breed: purebred cats have an increased risk
  
  • Loss of hybrid vigour
  • Indoor housing and multiple cats may increase the viral load

Question 26

Briefly describe the viral shedding of feline coronovirus and the relationship with the clinical disease FIP

Answer 26

• Virus can be shed as ealry as 2 days post infection
  
  • Replication is likely in the SI epithelial initially and then at the ileocolic junction for longer term shedding
• Type II are shed for a couple of weeks, whereas the type I virus may be shed for 2-3 months with ~13% of cats establishing a persistent infection
  
  • With a persistent infection, the same strain is typically shed in the faeces for life
    
    • RT PCR positivity for 9 months is required to diagnose persistent infection
    • Persistently infected cats do not develop FIP
• Serology negative cats do not shed the virus
• 1/3 seropositive cats will shed the virus, with higher titres correlated with the risk of shedding

Question 27

Discuss the transmission of feline coronavirus

Answer 27

• Most cats are infected by indirect faecal oral transmission
• Highly contagious virus with most cats in a multicat household likely to seroconvert
• The virus is readily inactivated by most household cleaners
  
  • Bleach is preferred as it is safe around cats
• Immediate post-natal infection can occur, but transplacental infection is unlikely

Question 28

Discuss the infection of host monocytes by feline coronavirus and how this contributes to the development of the disease FIP

Answer 28

• There is a variability of the host monocytes to be infected by FCoV. ie. it is not only the virulence of the virus that contributes
• FCoV infected monocytes in the blood stream extravasate into the interstitial tissue - this is the key event in the devlopment of FIP
  
  • The infected macrophages release IL-6, IL-1b, matrix metalloproteinase (MMP) and TNF-a

IL-6:

• In early infection, IL-6 stimulates hepatocytes to produce acute phase proteins - alpha-1 acid glycoprotein
• IL-6 stimulates the differentiation of B lymphocytes to plasma cells
  
  • IL-6 therefore contributes to the development of gammaglobulinemia

TNFa:

• Major contributor to the inflammatory response and contributes to lymphopenia
  
  • May contribute to lymphocyte apoptosis
• Upregulates the production of fAPN (receptor for type II FCoV), GM-CSF and G-CSF (both from macrophages) - these all contribute to neutrophil survival and neutrophilia
• TNF-a is also a major driver of cachexia

IL-1b:

• Pyrogenic and contributes to the inflammatory response

MMP 9:

• Zinc-dependent endopeptidase that is capable of breaking down the extracellular matrix
  
  • Probably contributes largely to the vasculitis and extravasation of fluid in effusive FIP

Question 29

Discuss how the type and strength of the immune response affects the course of disease following feline coronavirus infection

Answer 29

• Stress is common leading up to FIP presentation
  
  • Marked increase in viral shedding with stress
• Theory:
  
  • strong CMI response - no FIP
  • Weak CMI and strong humoral response - effusive FIP
  • Intermediate response - non-effusive FIP
• Greater survival with strong CMI response whereas a strong humoral response (against the spike protein) is required to clear the virus
• It is suspected that FCoV is able to suppress the immune system to allow persistent infection - low grade / subclinical infection but persistent shedding
• Higher viral levels in cats that succumb to FIP
• Host cells express viral antigen. The additional presence of antibodies on those cells cause internalisation of the Ag-Ab complex, delaying the clearance of the infected cells

Question 30

Describe the multisystemic vascular disease that causes effusive and non-effusive FIP

Answer 30

• The effusive and non-effusive forms of the disease are essentially a continuum of the same process of varying severity
• The inflammatory disease is essentially a pyogranumoatous vasculitis
  
  • There is diffuse inflammation of many blood vessels in the effusive form
  • There is patchy localised inflammation in the non-effusive form
• There is typically a long to very long incubation period, especially with the non-effusive form
• The disease is commonly seen in the weeks to months following a stressful event - immune suppression resulting in increased viral replication

Question 31

List the various organ systems affects and clinical signs that may be seen with the non-effusive form of FIP

Answer 31

Note: The presenting signs for a cat with non-effusive FIP can be vague and varied with not all signs present in all cats

1. Ocular signs
   
   • Iritis
   • Keratic precipitates
   • Retinal lesions - pyogranuloma, perivascular cuffing, haemorrhage
2. Neurological signs
   
   • Present in 25-33% of cats with noneffusive FIP
   • Variable and reflect the area of the CNS affected
     
     • Altered mental status
     • Ataxia - cerebellar, sensory or central peripheral
     • Seizures
   • Pyogranulomatous meningitis
   • Focal pyogranuloma on a peripheral or cranial nerve
3. Colonic / intestinal signs
   
   • Constipation, diarrhoea, vomiting
   • ​Seen primarily with an ileocolic localisation
4. Cutaneous lesions
   
   • Occasionally seen and other signs are always present
   • Non-pruritis, small 2 mm, round circumscribed , slightly raised papules

Question 32

List the available diagnostic tests for feline infectious peritonitis

Answer 32

• Serology
  
  • Detection of antibody titres
• Effusion analysis
  
  • Rivalta test - PPV = 0.97, NPV = 0.86
  • Basic fluid analysis - high protein transudate
    
    • Albumin globulin ratio in the fluid correlated to FIP
• Haematology and biochemistry
  
  • Lymphopenia with a mild non-regenerative anaemia
  • Polyclonal gamma-globulin increase
  • Elevated globulin, normal to low albumin
• CSF
  
  • Pleocytosis in 67% and elevated protein
  • Ag (PCR) or Ab limited clinical utility
• RT-PCR
  
  • Performed on tissue samples or effusion
  • Quantitative methods likely reduce the risk of a flase positive due to circulating FCoV in a cat with other disease
• Ag detection in tissue
  
  • Immunofluoresence, immunohistochemistry - identification of virus within the macrophages is considered the absolute gold standard
  • A negative control must be used
• Alpha-1 Acid Glycoprotein
  
  • Inflammatory marker produced by the liver under the influence of IL-6.
  • Used to help comfirm but not-specific

Question 33

Describe the indications, utility and limitations of serology in the diagnosis of feline infectious peritonitis

Answer 33

• Antibodies appear 18-21 days post-infection
• FCoV antibodies can be present in FIP and cats that have previously been exposed to FCoV and have or will clear the virus
• Negative results are more likely present in a cat free of the disease, however they can be negative in the presence of large viral loads due to binding
• Cats with non-effusive FIP usually have high FCoV titres, thus a low titre can be used to help exclude the disease
• Serological results CANNOT be used alone to confirm diagnosis
• Monitoring treatment - reduced Ab titre to zero indicates clearance of the infection
• High FCoV titres in effusion fluid have a 86% and 85% sensitivity and specificity for FIP respectively
  
  • A negative result can occur with high Ag:Ab complexes - RT PCR can help confirm this finding
• Screening:
  
  • Major reason for the test in cattery situation
  • Presence of the virus
  • Prior to mating
  • Prior to introductions
  • Prior to surgery
  • Prior to administration of immunosuppressive medications

Question 34

List and briefly comment on the treatment options for feline infectious peritonitis

Answer 34

• Corticosteroids
  
  • Immunosuppressive dose ~4 mg/kg for 10-14 days, then 2 mg/kg for 10-14 days, then 1 mg/kg for 10-14 days and so on
• Remdesivir
  
  • Nucleoside analogue
  • Blocks the action of reverse transciptase and viral replication

Question 35

Discuss the aetiology of Feline Leukemia Virus Infection

Answer 35

• FeLV is a gamma-retrovirus
  
  • Protein core - single stranded RNA within an envelope
• Replicates in many tissues including the salivary glands and respiratory epithelium and will eventually move to the bone marrow and infect the haematopoietic stem cells if the immune system does not clear the infection
• Reverse transcription of the RNA and random integration into the host DNA occurs
  
  • RNA is synthesized together with the viral proteins and assembly occurs near the cell membrane
• The new virions bud from the host cell membrane
• DNA incorporation does not usually result in cell death
  
  • Therefore, all daughter cells contain the viral provirus DNA
  • Once established in the haematologic and immune stem cells true elimination of the virus becomes impossible

Question 36

Briefly explain the presence of endogenous retrovirus virus in the feline genome.

How is the presence of endogenous FeLV relevant in FeLV infection?

Answer 36

• Retrovirus’s are single stranded RNA viruses that initially replicate by generation of a DNA strand via reverse transcriptase
  
  • This DNA is then randomly incorporated into the host cell DNA
  • There are segments of retovirus DNA that have been passed to offspring after they became benign and did not generate significant or deadly disease in the host
• FeLV A is passed from cat to cat and is an exogenous retrovirus
• FeLV B and FeLV C subgroups are generated when there is recombination of FeLV A subgroup virus with endogenous viral DNA in vivo
  
  • Both FeLV infection and host endogenous retroviral DNA must be present to form the B and C subgroups
• This recombination process may increase the pathogenicity

Question 37

What are the major FeLV subgroups?

Describe the pathogenicity and primary clinical outcome with each.

Answer 37

• FeLV has three major subgroups - A, B, C
• FeLV A is the only subgroup that undergoes horizontal transmission, while the others form in vivo by recombination after infection with the FeLV A subgroup
• Generally, the subgroups B and C are more highly pathogenic
• Subgroup B is most often associated with lymphoma
  
  • 100% of kittens developed lymphoma by 1 yer of age in an experimental study
  • Also been associated with the majority of cats that develop thymic (mediastinal) lymphoma
• Subgroup C is most often associated with fatal non-regenerative anaemia

Question 38

Briefly describe the structure of the FeLV genome

Note the product or effect from each of the various segments

Answer 38

• There are gag, pol-env gene sequences that are flanked by a long terminal chain (LTR) at each end

LTR:

• play a role in tissue tropism
• Contain enhancer sequences (upstream region enhancers - URE) that may play a role in oncogenesis
• FeLV U3-URE upregulates NF-kb

gag (group-associated antigen):

• Encodes for the internal structural proteins
• One of these, p27 is produced at vastly increased numbers from what is needed for new virus assembly
• p27 is the antigen detected by many ELISA tests

pol:

• encodes the RT-DNA polymerase

env:

• Encodes the envelope proteins and defines the subgroup
• Important for inducing immunity
• gp70 is important for natural resistance - antibodies to this protein result in virus neutralisation
• gp70 is a target for vaccine production

Question 39

Comment of the prevalence and change thereof for Feline Leukemia Virus.

Also note the breed, age and other characteristics that predispose to infection

Answer 39

• Prevalence has generally decreased over the past 30+ years
  
  • Test and removal programs by breeders is largely responsible
  • Reduced social and roaming behaviour has been associated with a reduced prevalence
• Currently, the prevalence is low in developed countries at <1 - 2%. May still be quite high in less developed countries
  
  • Multi-cat households and roaming / fighting may increase the risk/prevalence
• Pure bred cats less risk - likely due to breeder awareness and more likely to be kept indoors
• Regressive infection cannot be identified by Ag detection ELISA testing - may under-report true prevalence
  
  • Testing for Proviral DNA may improve the sensitivity of prevalence/screening studies

Question 40

Discuss the important and potential routes of FeLV transmission

Answer 40

• FeLV is primarily spread through direct contact with saliva
• Virus particles are at a higher concentration in saliva that they are in blood during viremia
• Transmission is almost uniformly cat to cat with minimal survival of the virus in the environment
• Both healthy and sick viremic cats will have high virus loads in the blood and saliva
• Small amount of viral DNA and RNA are excreted in faeces / urine, but infection via this route is unlikely
  
  • More likely to develop antibodies than infection
• Fleas can transmit viral DNA/RNA but this route is not considered significant
• Fomites including needles, and contaminated instruments or blood produces are a potential risk

Question 41

Explain the susceptibility to FeLV in kittens and the factors that increase or decrease the risk of a kitten devloping persistent viremia

Answer 41

• Susceptibility to becoming persistently viremic is highest in young kittens
  
  • Exposure is primarily from other cats in the environment
    
    • High density or known infected cats in the environment increase chances
    • Infected queen can transmit the virus transplacentally, via saliva or via milk
• Susceptibility likely starts to reduce by ~4 months of age
  
  • Possibly due to reductions in the number of cellular receptors necessary for FeLV to enter a target cell
  • Prevalence of natural antibodies gradually increases over time and with age
• Despite the age related decrease in susceptibility, there is still an increased risk if adults are housed with known viremic cats

Question 42

List the various stages / types of infection or outcomes that can result from FeLV infection

Answer 42

1. Abortive infection
2. Regressive infection
   
   • Can develop latent infection with immunosuppression or other disease
3. Progressive infection
4. Focal or atypical infection

Question 43

Describe what is meant by a regressive infection in terms of FeLV

Answer 43

• Regressive infection refers to a state where proviral DNA remains present and is incorporated into the host genome, but these cats do not produce active virus and are not viremic
• Regressive infection does not cause viremia, therefore these cats are unlikely to shed the virus
  
  • blood transfusion may be a transmission risk
• Newer research suggests that most cats remain infected for life in this regressive state

Question 44

Describe what is meant by an abortive infection with FeLV

How might this scenario occur and how likely is an abortive infection?

Answer 44

• Abortive infection refers to a complete and neutralising immune response that results in no viremia and no incorporation of proviral DNA into the genome
• For an abortive infection to occur, low level exposure is the likely trigger, with an adequate CMI response together with effective production of a humoral response
• Viral neuralising antibody titres tend to be high in these cats
• Persistence of high level antibody titres may be due to low levels of proviral DNA being present within the genome
  
  • ie. not truly an abortive infection but regressive

Question 45

Describe the process of development of regressive infection following FeLV exposure

Answer 45

• Viral exposure via the oral route
• Replication in the local lymphoid tissues and viremia develops
• Immune response matures and contains the viremia either immediately prior to or soon after bone marrow infection
  
  • Initial viremia causes clinical signs such as malaise, fever, lymphadenomegaly
• Viremia is terminated within 3-6 weeks in most cats
• Virus is encorporated into their genome, however, they have effective circulating antibodies and are protected against subsequent exposure
• If the viremia persists for long enough, the bone marrow and eventually the stem cells may become infected.
  
  • In this stage, the viral particles can be detected by immunofluorescence due to high viral loads in the platelets and granulocytes
• If present within the bone marrow, latent infection is established - a stage of regressive infection

Question 46

Describe the process that leads to development of a progressive FeLV infection

Answer 46

• Progressive FeLV infection is one in which there is a persistent viremia
• Infection occurs as normal via the oral route
• Viral replication occurs in the local lymphoid tissues with subsequent viremia and infection of the bone marrow together with mucosal and glandular epithelial tissues
  
  • An ineffective CMI and humoral response leads to ineffective clearance of the virus and low levels of neutralising antibody
• Proviral DNA has been incorporated into the stem cells of the bone marrow
• Virus is able to persistently replicate in the bone marrow, spleen, lymph nodes and salivary glands
• Most cats will succumb to an FeLV associated disease within 3 years
• The initial viral load is no different between progressive and regressive infection - the host immune response is the major difference

Question 47

What are the most likely clinical outcomes in a cat with progressive FeLV infection

Answer 47

1. Anaemia and haematological abnormalities
   
   • Primarily caused by subtype C
     
     • Interferes with a heme transport protein
   • Severe non-regenerative anaemia is most common
     
     • Regenerative anaemia can be seen with opportunistic Mycoplasma haemofelis
   • Direct suppression by the virus, chronic inflammatory disease and myeloproliferative disease may all contribute to anaemia
2. Bone marrow suppression
   
   • Complex and severe
   • Thymic atrophy, lymphopenia, neutropenia, impaired neutrophil function, loss of CD4⁺ and CD8⁺ cells
   • Secondary infection or exacerbation of infection is likely
3. Lymphoma / leukemia
   
   • Mostly associated with subtype B
     
     • Develops due to transduction or insertion of a cellular oncogene or acts to inactivate a suppressor
   • Activation of the oncogene can lead to uncontrolled proliferation
   • U3-LTR can upregulate NF-kb

Question 48

Note the potential mechanisms by which FeLV infection could contribute to myelosuppression

Answer 48

• Stimulation of neoplasia leading to myelophthis
• Development of myelodysplastic syndrome
• Myelofibrosis due to chronic inflammation or increased bone marrow activity
• Provirus may interrupt or inactivate cellular genes after being inserted into the cellular DNA
• Regulatory changes within the cell may affect neighbouring cells
• Cytokine production may be reduced or increased
• Stromal progenitor cells may functino inadequately to maintain the BM microenvironment
• Cellular expression of viral or abnormal antigens may trigger immune mediated destruction

Question 49

Describe the mechanism of action and potential platelet abnormalities seen with clinical FeLV infection

Answer 49

• Thrombocytopenia
  
  • Reduced life span
  • Decreased production in the bone marrow
    
    • primarily due to viral targeting of the megakaryocyte
    • As a component of pancytopenia
  • Immune mediated targeting in combination with IMHA - due to targeting of abnormal membrane proteins
• Thrombocytopathy
  
  • Giant platelets
  • Size, shape and function changes

Question 50

Describe the pathophysiological mechanisms that contribute to the various potential leukocyte abnormalities with FeLV infection

Answer 50

• Lymphopenia
  
  • Primarily due to viral replication
  • Thymus targeting contributes to preferential loss of T lymphocytes
• Neutropenia
  
  • Either mature neutropenia or myeloid hypoplasia with all granulocyte stages reduced
  • Immune mediated mechanisms likely as some will respond to glucocorticoid therapy
  • Maturation arrest can be seen in the bone marrow
  • Cyclic neutropenia described

Question 51

List and describe the pathophysiological mechanisms of the potential immune mediated diseases seen secondary to FeLV infection

Answer 51

1. Haemolytic anaemia
   
   • Due to expression of viral antigen on the red blood cells or dysregulation of the normal humoral response to self-antigen
2. Neutropenia
   
   • As for anaemia
3. Glomerulonephritis
   
   • Immune complex deposition - type 3 immune disease
   • Various antigens may contribute including p27, gp70, p15E and whole virus
4. Polyarthritis
   
   • as for glomerulonephritis
5. Uveitis
   
   • As for glomerulonephritis

Question 52

List the diagnostic tests available to investigate for FeLV infection when suspected

Describe the utility of each test

Answer 52

1. Viral detection
   
   • ELISA - detection via MAB adherance to the p27 antigen
     
     • Recommended screening test. False negatives do occur
   • Immunofluorescence - identify specific viral particles in the blood cells. Poor sensitivity and does not become positive until ~ 3 weeks after infection
     
     • Positive result likely reflects a cat that will develop progressive disease
2. Nucleic acid detection
   
   • Can be performed on blood, tissue, bone marrow or saliva
   • DNA provirus or viral RNA can also be detected
     
     • Can identify regressive infection, though presence of regressive infection does not prove causation
3. Antbody detection methods
   
   • Unhelpful except for potentially assessing the FeLV status of a population
   • Abortive or regressively infected cats will have high antibody titres whereas progressively infected cats may have no antibodies

Question 53

Describe the aetiology of feline immunodeficiency viral infection

Answer 53

• FIV is a lentivirus - retrovirus - encapsulated, single stranded RNA virus
• At least 5 subtypes / clades, with A and B predominating in Australia
• FIV has the usual env, pol and gag genes together with other accessory genes
• The different env gene properties affect cellular tropism and influence pathogenicity
• The env proteins are the target of the immune response
• Mutations of the viral genome and env gene during the course of infection could contribute to variants that resist the effect of neutralising antibodies

Question 54

Describe the routes of transmission of FIV.

Note the clinical importance of each

Answer 54

• Virus is readily present in saliva and blood
• Fighting, biting and wounds
  
  • Accounts for the increased prevalence in intact males
  • The most significant cause of spread
• In utero infection and postpartum transmission in milk
  
  • The virus can be concentrated in the milk
  • Both these routes are considered of minimal importance in natural infection
• Mucosal transfer can occur - intravaginal, intra-oral, intrarectal innoculation
  
  • CD4⁺ and CD8⁺ T cells, B cells, macrophages and dendritic cells can be infected transmucosal
    
    • These spread sytemically within days
• Provirus may be transmitted from the dam without FIV antibodies
• Horizontal transmission in a household is unlikely unless there is fighting behaviour

Question 55

List the various factors that interact and contribute to the pathogenesis of FIV infection

Answer 55

1. Age at the time of infection
2. FIV properties including subtype
3. The volume of virus particles innoculated
4. Route of infection
   
   • Parenteral more significant than transmucosal
5. Cell-associated or cell-free innoculum

Question 56

Describe the initial pathophysiological processes that occur following FIV innoculation / infection

Answer 56

• Following innoculation - by which ever route - virus is cleared by tissue macrophages
• Virus then replicates in the local and distant lymphoid tissues - LN, spleen, thymus
• Viremia is readily detected by PCR within 2 weeks of infection and persists
• Virus can also spread to the monnonuclear cells in many organs including the brain, kidney, BM, lung and GIT
• A humoural immune response can be detected by 2-4 weeks post-infection and is usually vigorous
  
  • Antibodies are diverse and neutralising antibodies can be produced
  • Neutralising antibodies do not effectively target the intracellular virus
  • Antibodies are unable to affect the proviral genes that are inserted into the host cells
• A CD8⁺ CMI response may be more important than the humoral response at clearing the initial viremia
  
  • Production of soluble factors that likely inhibit viral messenger RNA transcription

Question 57

Describe the pathophysiological processes that occur following the initial viremia and humoral immune response

Answer 57

• Following the initial viremia, cats enter a clinically asymptomatic period
• Minimal virus is produced, but this is not true latency
  
  • Virus can still be recovered in blood lymphocytes, CSF, semen, serum/plasma and lymphoid tissues
• Plasma levels of virus particles can increase again in the terminal phase of infection
  
  • Likely associated with a decrease in the CD4⁺ T cell number
    
    • Due to apoptosis, decreased production, lysis due to the virus itself or destruction of the virally infected cells by the immune system itself

Question 58

Describe briefly the other (over and above CD4⁺ loss) immune dysfunctions that occur over time with FIV infection

Answer 58

• Variable and altered tissue cytokine profiles
  
  • INcrease INF-y, TNF-a, IL-4, -6, -10, -12
• Reduced ability for lymphocytes to proliferate
• Impaired lymphocyte priming by immunogens, mitogens or recall antigens
• Due to the altered immune response, other infections can occur or persist
  
  • Toxoplasmosis
• Impaired neutrophil adhesion and emigration
• Diminished NK-T cell activity
• Hypergammaglobulinemia and delayed class shift from IgM to IgG

Question 59

Briefly describe the clinical findings in cats with FIV infection

Answer 59

• The initial infection and viraemia typically causes mild and non-specific signs only
  
  • Fever, malaise, enteritis, stomatitis, conjunctivitis
  • Lymphadenopathy is common
• Following the inital viremia, clinical signs are typically absent for months to years
• With progressive disease, the clinical signs typically relate to the signs from secondary disease
  
  • Opportunistic infection including toxoplasmosis, fungal infectionm mycoplasma, bartonella, other viral infections
  • While the majority of these infections are no more prevalent in the FIV infected population, the associated clinical signs can be worse due to immunosuppression / immune dysregulation
• Development of lymphoma or leukemia
  
  • Likely indirect cause as FIV proviral DNA is only rarely identified in the neoplastic cells

Question 60

List the available tests to investigate a suspected FIV infection or screen for FIV infection in a cats

Note the indications and limitations of each

Answer 60

1. Antibody testing
   
   • ELISA or immunomigration assay
   • High sensitivity and specificity - good screening test
   • Most infected cats will readily and continually produce antibody
   • Delay in antibody production for 2+ months may account for early negative results
   • MDA can cause positive results in kittens
   • Vaccine derived antibodies will cause a positive result
2. Virus detection
   
   • PCR methods to identify proviral DNA
   • Typically a highly sensitive testing modality and false positives may occur
   • Due to variable subtypes, individual PCR methods may not identify all variants - false negatives
     
     • Sensitivity variable from 41-93%
     • Specificity has ranged from 81-100%