Canine parvovirus: An update on variants

Q: Please review what's happening with canine parvovirus in the United States.

A: Dr. Sanjay Kapil gave an excellent lecture on "Canine parvovirus variants circulating in the United States (2006-2008)" at this year's American College of Veterinary Internal Medicine Forum in Montreal, Canada. Here are some relevant points from it:

Canine parvovirus (CPV) emerged as a new pathogen of dogs in the 1970s. Today, it is the most common viral disease of dogs. There are two canine parvoviruses: canine parvovirus-1 (minute virus of canines) and canine parvovirus-2, the primary cause of puppy enteritis, morbidity and mortality. CPV-1 and CPV-2 are antigenically and genetically different.

Canine parvovirus contains a single-stranded DNA of about 5,000 bases. This small virus has only major proteins, viral protein 1 (VP1) and viral protein 2 (VP2). CPV is a non-enveloped virus that is resistant to lipid solvents, temperature and pH changes. Due to a compact DNA genome, parvovirus has high physical resistance.

CPV-2 can survive in the kennel environment for months or years. If the kennel is not properly cleaned, the virus can be transmitted to the next batch of naïve puppies.

Canine parvovirus genome tends to mutate at critical amino acids (about 10 residues) of VP-2 capsid protein.

Evolution of CPV-2 in the last 30 years

A CPV-2 pandemic began in 1977. It was associated with high mortality due to hemorrhagic diarrhea and myocarditis. The virus probably originated from a carnivore parvovirus (feline panleukopenia, mink parvovirus or blue fox parvovirus).

These viruses have high genetic similarity (99 percent). However, they have biological differences, such as differences in the suitable pH for maximum hemagglutination activity. For example, based on a recent study, feline parvovirus interacts with transferrin receptor at a pH of 6.5. However, a recent CPV-2c can interact with transferrin from pH (3-8).

These genetically closely related viruses have acquired host-range differences due to single-point mutations on the VP2 protein. The prototype CPV-2 isolates did not infect cats but could grow in feline cell lines such as Crandall Reese feline kidney (CRFK) cell line. CPV-2 soon acquired the ability to infect cats by point mutations that broadened the host range.

By 1982, a genetic variant emerged that had amino-acid changes at positions 87, 101, 300, 305 and 555 of the VP2 protein. This genetic variant was designated CPV-2a to distinguish it from the original CPV-2. CPV-2a had acquired the ability to infect cats, and it had isoleucine instead of valine at position 555 of the VP2 protein.

In 1985, another genetic variant designated CPV-2b emerged, spreading worldwide. It is the major genotype of CPV-2 in the United States. In 2000, Dr. Buonavoglia from Bari, Italy, reported a new genotype of CPV-2 that had glutamic acid at position 426 of the VP2. This virus is the second major genotype of CPV-2 in the United States. CPV-2c has now been documented on all continents except Australia. In addition to natural evolution of CPV-2, there are other mechanisms of antigenic and genetic changes in the current CPV-2 isolates.

Diagnosis

Canine parvovirus is the most common cause of enteric disease in puppies. After oral infection followed by systemic infection, canine parvovirus is shed for five to seven days. However, it has been shown that dogs infected with CPV-2c may shed the virus for up to 51 days.

There are a variety of tests available for laboratory-based diagnosis of CPV-2 infection. For ante-mortem diagnosis, fecal samples (2-5 grams) are preferred. For post-mortem diagnosis, pieces of unfixed, small intestine and tongue are preferred.

Canine parvovirus can cause focal infection of the intestines. Three discontinuous loops of the small intestine should be submitted for direct fluorescent antibody testing. Anti-CPV-2 monoclonal antibody 3B10 from VMRD Inc. has been found to react with all circulating genotypes of CPV-2 in the United States. This antibody is suitable for immunohistochemistry on formalin-fixed tissues.

For genotyping, nucleic acid-based techniques are needed. CPV-PCR can be used and followed by sequencing and analysis of the sequence. Amino-acid position 426 determines the genotype of the CPV-2. For CPV-2 and CPV-2a, the amino acid at position 426 is asparagine (Asn); for CPV-2b, the 426 is aspartic acid (Asp); and for CPV-2c the amino acid at position 426 is glutamine (Glu).

In the clinic, ante-mortem diagnosis can be performed using ELISA and SNAP tests. A variety of factors, including the time of collection of the samples after infection, can affect the outcome of these diagnostic tests.

All CPV-2 viruses hemagglutinate erythrocytes. This universal property of the virus can be used to develop an instant, easy, robust animal-side diagnostic test for CPV-2 antigen (SAT) and antibody diagnosis (SIT).

These instant tests do not require any instrumentation and are suitable for application in kennels by owners because they require minimal training. The procedure requires mixing a drop of fecal sample and erythrocytes (porcine erythrocytes or canine and feline erythrocytes). The agglutination reaction is quick/instant. The test performs well at room temperature, and with ll circulating genotypes of CPV-2 in the United States.

Vaccines

Current CPV-2 vaccines can be classified according to the amount of CPV-2 antigen: high- and low-antigen-dose CPV-2 vaccines. High-antigen-dose vaccines claim to immunize the puppies in the presence of maternal antibodies. Higher amounts of CPV-2 vaccine will lead to higher and longer shedding by the puppies, leading to second-hand exposure to the virus. Most major commercial vaccines contain modified-live virus (MLV) of CPV-2 or CPV-2b genotypes. There is no commercial vaccine that contains CPV-2a. Moreover, CPV-2a has not been found in nature in the United States.

Commercial vaccines containing CPV-2 isolates may not provide robust serum antibody responses against CPV-2b and CPV-2c isolates. It was recently published that all commercial vaccines provide protection against current circulating genotypes of CPV-2 based on experimental studies just presented recently. However, other published studies contradict claims of CPV-2 vaccine efficacy under field conditions.

Clearly, the observations made by diagnostic virologists/pathologists, canine breeders, field veterinarians and shelters do not agree with the universal efficacy of CPV-2 vaccines worldwide. After the introduction of commercial CPV-2 vaccines, the problem is significantly reduced but CPV-2 continues to be the No. 1 infection in puppies, worldwide.

CPV-2b vaccines have been found to have higher efficacy compared to some available CPV-2, based on the published literature. Currently, there is no commercial vaccine that contains CPV-2c antigen. Commercial companies are evaluating the efficacy of MLV CPV-2c vaccines.

Antigenic cross-reactivity

Serum antibody titers are a good measure of immune status of the dog against CPV-2.

Antibodies against one genotype will cross-react with other genotypes of CPV-2. Traditionally, hemagglutination-inhibition tests using porcine erythrocytes is used to assess the level of antibodies in serum samples. A titer of > 1:80 is considered to be protective. Most commercial vaccines induce this titer after a single vaccination in puppies that are serologically negative.

Serum neutralization testing is slightly cumbersome, but it is the preferred method for determination of the animal's immune status. Differences in SN titers among CPV-2 isolates are considered insignificant if it is < 4-fold SN titers. CPV-2 isolates that differ by 4-fold or more are considered significant. Anti-CPV-2c antiserum reacts better with CPV-2c and CPV-2b isolates compared with CPV-2b antiserum.

Vaccine failure

Under field conditions, vaccine failures happen. The most common reason is the incomplete take of the MLV vaccine due to interference from maternal antibodies. This problem is exacerbated due to less efficient neutralization of the variant CPV-2 isolates that have emerged recently.

Other factors, such as biological variation among CPV-2 isolates, may be responsible for the recent CPV-2c problems. In the last three years, the frequency of GCA at position 440 has increased in the CPV-2c isolates. The CPV-2c and CPV-2b isolates recovered from puppies older than 5 months have a higher frequency of GCA at position 440 compared to ACA among CPV-2c and CPV-2b isolates recovered from puppies younger than 5 months.

Disinfection

Canine parvovirus is resistant to most common disinfectants. Household bleach (1:30 dilution in water) and potassium peroxide (Trifectant or Virkon) are suitable for all CPV-2 outbreaks. Ten minutes of exposure to these chemicals will destroy CPV-2 virus. All canine parvovirus isolates have similar properties. Items that cannot be treated with either of these chemicals can be disinfected with steam.

Geographic distribution

To date, CPV-2c has been detected in samples from a total of 15 states. Unrestricted movement of infected dogs has been responsible for transatlantic transmission of CPV-2c from Italy. Highest numbers of CPV-2c cases have been detected in Oklahoma. The geographic and regional monitoring is important so that proper genotypes can be selected for vaccine production in different countries of the world. Moreover, it will be important to understand how to introduce the vaccines with different genotypes in different U.S. states. It will be prudent to continue using the current MLV CPV-2 vaccines for most of the states and to introduce CPV-2c as soon as it becomes available.

Effects on wildlife

Canine parvovirus is a major threat to conservation of wolves. About half of the wolf puppies in Minnesota have succumbed to canine parvovirus. Carnivore parvovirus isolates have caused disease in Lynx, bobcats and raccoons. There are few studies in which the wildlife parvovirus isolates have been characterized. Most have used serology to detect the exposure.

Conclusions

Canine parvovirus emerged about 30 years ago. The virus has spread rapidly across continents, undergoing rapid evolution in adapting to the new host. Due to movement of dogs, CPV-2c has spread worldwide and CPV-2c has become the major genetic variant in Europe.

In the United States, CPV-2c is accounting for more cases of canine parvovirus infection. CPV-2c cannot be clinically distinguished from other CPV-2 genotypes. CPV-2c is mostly a disease of young puppies. There is a debate over whether the canine parvovirus-2 vaccines need to be updated.

Several groups, including vaccine companies, have shown that current vaccines provide protection against the Italian CPV-2c isolate under experimental conditions in sero-negative dogs. Based on large numbers of independent reports from dog breeders in the south-central states, current vaccines need to be carefully evaluated for their efficacy against current circulating CPV-2 isolates.

At least three independent published reports suggest that CPV-2 isolates can differ in their biological properties and CPV-2 isolates (about 30 percent) can have significant antigenic differences (about 10-fold). This level of antigenic distance is significant and will make a difference in puppies that are maternal antibody-positive at 6 weeks of age.

It has been recommended to give a booster shot to puppies at 4 months of age to cover the emerging CPV-2c isolates. This additional booster vaccine will help increase the number of puppies (population immunity) that have actively seroconverted before they are put in the CPV-2 contaminated environment.

by Johnny D. Hoskins DVM, PhD, Dipl. ACVIM

Dr. Hoskins is owner of Docu-Tech Services. He is a diplomate of the American College of Veterinary Internal Medicine with specialities in small animal pediatrics. He can be reached at (225) 955-3252, fax: (214) 242-2200 or e-mail: jdhoskins@mindspring.com