Virulence In Ferrets Research Articles

Immunohistochemical analysis of two strains of lion (Panthera leo)-adapted canine distemper virus in ferrets (Mustela putorius furo).

Canine distemper virus (CDV) caused epizootics in lions (Panthera leo) in Tanzania's Serengeti National Park in 1994 and in captive lions and other Panthera spp. in the USA in 1991-1992. In this study, immunohistochemistry was used to compare viral distribution in tissues collected from ferrets (Mustela putorius furo) inoculated with one of the two lion-derived CDV isolates, either from Serengeti (A94-11/13) or from California (A92-27/20). The California isolate resulted in severe morbidity in all nine ferrets, whereas the Serengeti isolate resulted in severe morbidity in five of the nine ferrets. A slightly higher proportion of infected cells was found in many tissues in the Serengeti isolate-inoculated ferrets. These findings indicate that the pathogenicity of the California isolate is not directly related to the number of infected cells.

Nucleotide sequence and polymerase chain reaction/restriction fragment length polymorphism analyses of Aleutian disease virus in ferrets in Japan.

Two ferrets with spontaneous Aleutian disease (AD) were found in Japan. The diagnosis was verified by polymerase chain reaction (PCR) amplification of part of the capsid gene specific to AD virus (ADV). The nucleotide sequences (365 bp in length) of the amplified fragments from the 2 ferrets differed by a single nucleotide, producing an amino acid alteration. Compared with other types of ADV, these isolates had 96% sequence similarity to a published ferret ADV (FADV) in contrast to <91% homology to various types of mink ADV (MADV). The phylogenetic tree of ADVs indicates that these 2 isolates and the published FADV belong to the same genetic group and definitely are divergent from MADVs. The predicted amino acid sequence of the hypervariable segment in the capsid gene was conserved among the 3 types of FADV. These results indicated that the 2 isolates found in Japan were new DNA types of FADV and could have been derived from FADV(s). A restriction fragment length polymorphism (RFLP) method to distinguish the ferret types of ADV from the mink types of ADV was developed on the basis of differences in their nucleotide sequences. Digestion of the PCR products with Afal or ScaI provided different cleavage patterns for FADV and MADV. This PCR/RFLP analysis of the ADV capsid gene will be a valuable asset for diagnosis of this virus infection in ferrets.

GG167 (4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid) is a potent inhibitor of influenza virus in ferrets

GG167 (4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid) is a novel viral neuraminidase (sialidase) inhibitor which, following intranasal administration in ferrets, is at least 100 to 1,000 times more effective than ribavirin and amantadine against influenza A and B viruses. It retains its activity even when treatments are delayed until 24 h postinfection and has no effect on the serum antibody response to infection.

Efficacy of 2'-deoxy-2'-fluororibosides against influenza A and B viruses in ferrets.

Single-dose treatments (5 to 40 mg/kg of body weight given intraperitoneally) of ferrets with 2'-deoxy-2'-fluoroguanosine or its prodrug, 2,6-diamino-purine-2'-fluororiboside, 1 h after infection with influenza A virus significantly inhibited replication of virus in the upper respiratory tract, resulting in amelioration of fever and nasal inflammation. Replication of virus in the lower respiratory tract was also reduced > 100-fold, but three doses were required to prevent replication in the lungs. In ferrets infected with influenza B virus, single-dose treatment (40 mg/kg given intraperitoneally) produced a similar but reduced response in comparison with that in ferrets infected with influenza A virus, indicating that dosing was not optimal for this virus.

Virulence of Rimantadine-Resistant Human Influenza A (H3N2) Viruses in Ferrets

The influence of rimantadine-resistance mutations on the virulence of human H3N2 viruses in ferrets was examined. The similarities in virulence of the drug-resistant mutants with single amino acid substitutions at three different locations, 27, 30, and 31, within the M2 sequence and their corresponding sensitive wild-type isolates contrasted with differences in virulence between the three pairs of viruses. These data provide further evidence that rimantadine-resistant viruses that emerge during treatment of patients with the drug are unaltered both in their growth characteristics and virulence.

A mutation in the PA protein gene of cold-adapted B/Ann Arbor/1/66 influenza virus associated with reversion of temperature sensitivity and attenuated virulence.

Reassortant SG3 inherits only the acidic polymerase (PA) protein gene from the cold-adapted B/AA/1/66 influenza virus (ca B/AA/1/66) and all remaining genes from a virulent, wild-type virus. This reassortant demonstrates attenuated virulence in ferrets and expresses a ts phenotype characteristic of the ca parent. During virulence evaluation of SG3, a virulent, non-ts revertant virus (designated SG3rFL) was isolated from the lungs of one ferret. In order to determine whether the reversion of SG3 resulted from mutation of the PA gene and/or as the result of extragenic supressor mutations, the revertant PA gene of SG3rFL was transferred to a reassortant (SG3r) inheriting only the revertant PA gene from SG3rFL and all remaining genes from SG3. Reassortant SG3r was non-ts and virulent, indicating that mutation of the PA gene was sufficient for the reversion of the ts and attenuation phenotypes expressed by SG3rFL. The nucleotide and predicted amino acid sequences of the SG3rFL PA gene were determined and compared to those of wt and ca B/AA/1/66. The predicted PA proteins of wt and ca B/AA/1/66 are known to differ by six amino acid substitutions including a valine to methionine substitution at residue 431. The PA proteins of ca B/AA/1/66 and SG3rFL were distinguished by only the single amino acid substitution of methionine to isoluecine also occurring at residue 431. Thus, the methionine residue was implicated in the attenuation of ca B/AA/1/66 and its reassortants. The hydropathic properties of valine, isoleucine, and methionine suggested that reversion involved the restoration of hydrophobic character at this site.

Intestinal replication of influenza A viruses in two mammalian species. Brief report.

The sites of replication of influenza A viruses in ferrets and pigs were studied. The majority of the swine, equine, and avian influenza A viruses tested were recovered from the intestinal tract of ferrets as well as from the respiratory tract; most of the human influenza viruses studied were recovered only from the respiratory tract. In contrast with ferrets, only Hong Kong/1/68 (H 3 N 2) influenza virus was recovered from the intestinal tract of pigs. Despite the large biological variability found in ferrets and in pigs, the results do establish that the majority of influenza viruses have the potential to replicate in the intestinal tissues of some mammals. Additionally, the study suggests that there are differences among the influenza A viruses in tissue tropism in different mammals. Both viral and host genetic factors determine the tissue tropism of influenza viruses in mammals.

Attenuation of wild-type human influenza A virus by acquisition of the PA polymerase and matrix protein genes of influenza A/Ann Arbor/6/60 cold-adapted donor virus.

Wild-type influenza A viruses can be attenuated for humans by the acquisition of genes from the A/Ann Arbor/6/60 cold-adapted (ca) donor virus. Six-gene reassortants, that is, viruses containing the hemagglutinin and neuraminidase surface glycoprotein genes of the wild-type virus and the six remaining RNA segments of the ca donor virus, are consistently attenuated for humans. During the production of a six-gene reassortant virus containing the surface glycoproteins of the A/Washington/897/80 (H3N2) wild-type virus, a reassortant virus was isolated that contained RNA segments 3 (coding for the polymerase PA protein) and 7 (coding for matrix [M] proteins) from the ca parent and all other genes from the wild-type virus. This reassortant virus is referred to as a two-gene reassortant. Because the gene or set of genes responsible for the attenuation of ca reassortant viruses has not been defined, we evaluated the two-gene reassortant for level of replication and level of virulence in ferrets and in humans, and we compared its characteristics to those of a six-gene reassortant virus derived from the same two parents. The two-gene reassortant virus infected each of 14 adult seronegative (serum hemagglutination inhibition titer of less than or equal to 1:8) volunteers when administered intranasally at a dose of 10(7) 50% tissue culture infectious doses, yet it did not produce illness. The level of replication of the two-gene reassortant virus in the upper respiratory tract was equivalent to that of the six-gene reassortant virus. This demonstrates that transfer of the A/Ann Arbor/6/60 ca PA polymerase and M genes is sufficient to confer the attenuation phenotype on wild-type influenza A viruses. In the context of previous observations, these results suggest that the A/Ann Arbor/6/60 ca donor virus PA polymerase gene plays a major role in the attenuation of ca reassortant viruses.

Interference following dual inoculation with influenza A (H3N2) and (H1N1) viruses in ferrets and volunteers.

The effects of simultaneous inoculation with two attenuated influenza A viruses was studied in ferrets and volunteers. Groups of ferrets were inoculated with an influenza A (H3N2) or (H1N1), virus or a combination of both viruses: the temperature response, serum and local antibody response, and the change in nasal wash protein concentration was determined. The results showed that both viruses were attenuated for ferrets, and that inoculation with both viruses together did not cause clinical reactions. Serological studies on paired serum samples obtained from ferrets showed that both viruses when given separately infected all the inoculated animals; however, dual infection resulted in all ferrets being infected with the influenza A (H3N2) virus strain, but this infection interfered with infection by the influenza A (H1N1) strain. Similar investigations were carried out in volunteers. Again, the clinical reactions and temperature response of volunteers to infection by one or other of the viruses showed both strains to be attenuated for man even when given together. In addition, no adverse clinical reactions were seen in volunteers inoculated with both viruses simultaneously. Serum antibody studies showed that infection by influenza A (H1N1) virus interfered with infection by the influenza A (H2N2) virus strain. These results show evidence of interference by influenza A viruses; however, the direction of interference was one-way, and differed for ferrets and for volunteers.

Heterotypic immunity to influenza in ferrets.

Heterotypic immunity to influenza virus in ferrets operated against heterotypic influenza viruses but not heterologous viruses. Contrary to prior reports, the protection conferred lasted for at least 18 months. This type of immunity limited virus shedding but did not prevent infection. These results suggest that this phenomenon could play a role in determining the severity of infections caused by type A influenza viruses in humans.

Aspects of virulence in ferrets exhibited by influenza virus recombinants of known genetic constitution.

The parent viruses and clones 64c and 6 of the A/Puerto Rico/8/34-A/England/939/69 (H3N2) series were examined. Virulence was assessed from 50% infectious dose, replication in the upper respiratory tract, degree of lung infection, and production of fever. Viral infection of the upper respiratory tract declined with onset of the inflammatory response and pyrexia, and restriction of replication in nasal turbinate tissue at pyrexial temperatures correlated with onset and rapidity of decline of infection in the upper respiratory tract. Compared with the H3N2 strains, the HON1 strain of A/Puerto Rico/8/34 stimulated a lower and later inflammatory response, which correlated with a delayed and low fever and, in turn, a high nasal infection that was slow to decline. The differential ability to infect the lung in vivo (clones 64c and 6 more than A/England/ 939/69 and clone 7a more than A/Puerto Rico/8/34 and clone 64d) was not always explained by differences in ability to replicate in lung tissue at normal or pyrexial temperatures. Neither overall virulence nor any particular facet of it can yet be correlated with particular gene pieces. The results are discussed in relation to the virulence of the strains for humans.

The search for the ideal influenza vaccine.

The history of the development of influenza virus vaccine is traced from its origin with experimental studies of influenza virus in ferrets and mice and the first trials in man. Knowledge of the basis of immunity to the viruses in experimental animals and in man has grown steadily over the years and has been essential to successful immunization. Virus variation affecting the surface antigens of the virus is seen as the principal obstacle to the application of vaccines in man. So significant are the changes occurring during antigenic drift that former concepts of a polyvalent vaccine cannot provide a solution of the problem of the composition of vaccines. Disrupted virus vaccines appear to provide the answer to the prevention of vaccine reactions.

Contact transmission of distemper virus in ferrets

Distemper virus was transmitted when infected donor ferrets were placed with susceptible ferrets for various contact periods. Distemper was more likely to be transmitted during the later stages of the disease. A positive correlation was found between the length of contact time and the acquisition of infection.

Protective efficacy of RIT 4025, a live attenuated influenza vaccine strain, and evaluation of heterotypic immunity to influenza A viruses in ferrets

Ferrets immunized with an H3N2 recombinant of A/PR/8/34 and A/Scotland/840/74 (RIT 4025 vaccine strain) were almost completely protected against a challenge with the homologous strain A/Scotland/840/74. The protection was lower but highly significant when the challenge was performed with the heterologous A/Victoria/3/75 wild strain. The protection afforded by the vaccine strain was measured by three indicators: absence of temperature rise, absence or reduction of virus shedding and absence or reduction of nasal protein increase when compared with uninoculated controls. Heterotypic immunity in this animal model was not significant when these three indicators were measured after a challenge inoculation performed 5 weeks after immunization.

Growth of measles virus in nervous tissues. III. Neurovirulence of SSPE virus in ferrets.

The Niigata-1 strain isolated from a patient with subacute sclerosing panencephalitis (SSPE) was inoculated intracerebrally into ferrets. Neurological signs developed in about 1 week in most of the animals. Histopathological examinations of the central nervous tissues revealed degenerative lesions in the parenchyma of the brains and inflammatory reactions predominantly in the meninges and choroid plexus. Virus antigen was demonstrated mainly in the nerve cells by immunofluorescent staining. The results indicated high affinity of the Niigata-1 strain to the nerve cells. In contrast, the Mantooth strain of SSPE virus in cell-free state did not exhibit neurovirulence in ferrets.

Isoprinosine: lack of antiviral activity in experimental model infections.

This report is a condensation of data from a collaborative study proposed and sponsored by the Antiviral Substances Program of NIAID. The individual reports were prepared by the following persons: S. Baron and M. Worthington, NIAID, NIH; A. Friedman-Kien, New York University College of Medicine; J. C. Duenwald, Washington State University, L. A. Glasgow, M. Harmon, B. Janis, E. Kern, J. C. Overall, Jr., C. B. Smith, D. A. Stringfellow, and S. Westerberg, University of Utah College of Medicine; B. C. Easterday and E. H. Weinberg, University of Wisconsin.*Recently there has been much interest in isoprinosine as a broad-spectrum antiviral compound. The activity of this substance was evaluated in a coordinated study at five institutions. Experimental models in five species of animals were established using 11 viruses. Criteria for selection were: (l) representation of most major groups of viruses, (2) reproduction of natural routes of infection, and (3) simulation of potentially treatable viral infections of man. No therapeutic effect could be demonstrated in infections with encephalomyocarditis virus, type 2 Herpesvirus hominis, influenza, and rabies viruses in mice; vaccinia virus in rabbits; rhinotracheitis and panleukopenia viruses in cats; distemper virus in ferrets; and influenza and transmissible gastroenteritis viruses in swine. The only antiviral activity observed in this extensive series of experiments was suppression of fibroma virus lesions in rabbits given 600 mg/ kg per day of isoprinosine. Although antiviral activity is not precluded in other viral infections in animals or in man, these results clearly fail to substantiate the potential of isoprinosine as a potent, broad-spectrum antiviral substance.

Tissue Tropism of a Strain of Influenza a Virus in Ferrets

Summary The study dealt with the occurrence of febrile reactions and with the multiplication of virus in different tissues of the respiratory tracts of ferrets following infection with the Cam strain of influenza A virus. All animals were infected by the intranasal route while under anesthesia. No evidence for multiplication of the virus in the lungs was obtained either during ten serial passages in ferret nasal mucosa, or during five passages in ferret lungs; the latter procedure was made possible by alternate passages in eggs. The results as a whole indicate that at least some strains of influenza A virus are non-pneumotropic agents in ferrets. The results also suggest that fever produced in ferrets by the intranasal inoculation of large amounts of influenza virus is related to the toxic properties of the virus particles rather than to processes involved in multiplication of the virus.

Antibody response against strains of influenza-A virus in ferrets with basic immunity

In Holland ferrets are offered for sale with basic antibodies against one or more sub-groups of strains of influenza-A virus. With the haemagglutination inhibition test low basic antibody titers can only be detected by neutralising the non-specific inhibitor in the ferret sera. When such a ferret is infected with a strain of a heterologous sub-group, a considerable rise in antibodies is obtained against the homologous and heterologous sub-groups. It is very probable that the same holds for human beings. Some examples are given of haemagglutination-inhibition tests with serum pairs from adults who suffered from influenza in the epidemic of the winter of 1949 in Holland from which the non-specific inhibitor had been eliminated.