Date: Tue, 06 Feb 1996 10:22:37 -0400 (EDT)
From: MATSON@PICARD.EVMS.EDU


RABBIT VIRAL HEMORRHAGIC DISEASES (4)
======================================

I originally learned about the October release/escape of the rabbit
hemorrhagic disease calicivirus from the CSIRO study site through a
forwarded PROMED posting. I thought I would inquire through PROMED
of additional information about this event as I have been drawn into
the public discussion of the matter.

These are some of my concerns:

1.  Caliciviruses fall into five genomic groups by genomic
    organization and degree of shared sequence identity, as follows:

Group                           Known Hosts     Examples
---------------------
Small round structured viruses  Humans          Norwalk, Snow Mountain,
                                                and Hawaii viruses
Hepatitis E virus               Humans          hepatitis E strains of
                                                Burma, Mexico, India,
                                                Muslim states of former
                                                USSR
Rabbit CV-like                  Rabbits         rabbit hemorrhagic disease
                                                virus of China, Germany,
                                                Spain; European brown hare
                                                syndrome virus
Sapporo-like                    Humans          Sapporo calicivirus/82/
                                                Japan; Sapporo calicivirus/
                                                86/Houston; Sapporo
                                                calicivirus/90/Houston;
                                                Manchester and Plymouth
                                                viruses
Feline calicivirus-like      Cats, chimpanzee,  FCV F4, FCV-CFI, FCV-F9,
                            sea lion, dolphin,  Pan-1, Tur-1,SMSVs 1, 4,
                            mussel, sea otter,  13, 15, 17
                      Aruba Island rattlesnake
------------------------------------------------------------------------

The assortment of strains using phylogenetic methods based upon sequence
analysis and the assortment of strains according to genomic organization
resulted in identical clustering of strains. The concordance of the two
assortment methods brings a high level of certainty to the results. The
best way for me to show you this is to FAX interested individuals two
figures. If you can provide me a FAX number, the figures will follow
quickly.

Conclusions from phylogenetic analyses that led to the assortment into
five groups include:

a.  Small round structured viruses (SRSVs) are distinct and distant from
    other groups.
b.  Sapporo-like human caliciviruses cluster more closely to rabbit CV-like
    and FCV-like animal CVs than to hepatitis E or SRSVs.
c.  The FCV-like animal CV group includes strains of marine and terrestrial
    origin, establishing the hypothesis made 20 years ago that there is/has
    been movement of caliciviruses between marine and terrestrial reservoirs/
    hosts.
d.  The "proximity" of the Sapporo-like human caliciviruses and the two
    animal CV groups indicates that Sapporo-like CVs are/have been transmitted
    between animals and humans. Whether such transmission occurs now or
    remotely in the past is uncertain.
e.  The primate CV, Pan-1, lies firmly within the FCV-like group. This means
    members of four of the five known CV groups have infected primates.
f.  SMSV-5, a member of the FCV-like group, caused infection in a laboratory
    worker with manifestations of illness like those seen in the original sea
    lion host. This means four of the five known CV groups have infected
    humans.
g.  The only group not yet found in humans is the rabbit CV-like strains.
h.  The genome of the Sapporo-like CVs forms a superset for the rabbit CV-like
    and hepatitis E caliciviruses, with respect to genome organization. This
    is most clear in the figure I will FAX to you. By this, I mean that
    Sapporo-like HuCVs have the nonstructural polyprotein and the capsid gene
    in the same frame, probably capable of being synthesized in one long
    polyprotein. At the beginning of the capsid gene is another predicted
    protein product that overlaps the capsid gene, in another frame. The
    function of the protein encoded by this gene is unknown, but we know, at
    least for hepatitis E virus, that antibody to a protein encoded by a gene
    at this location is made in natural infections. Another predicted protein
    is located after the capsid gene. Sapporo-like HuCVs have all four of
    these genes, rabbit-like AnCVs have three of them, and hepatitis E virus
    has a different complement of three. The presence of "shared" genes does
    not imply that the functions of those genes are the same. However, the
    presence of them and their location requires common mechanisms for their
    expression during replication. The conclusion is, then, that the Sapporo-
    like HuCVs provide at least a biologic link of sorts between the rabbit
    and hepatitis E caliciviruses.

2.  The CSIRO has expressed confidence that there have been no reports of
    infection of humans by the rabbit hemorrhagic disease calicivirus during
    greater than 10 years of observation in sites where RHDV occurs. I think
    that experience is valid and worthy of full consideration. My caveats are:
    Has anybody looked for evidence of human infection in humans, such as
    performing serosurveys or targeted studies of rabbit handlers, foresters,
    game wardens, veterinarians, zoo workers, to see if they have antibody? We
    know CVs cross species boundaries, but does RHDV? Human disease may be
    absent, but infection might not be.

3.  Although CSIRO has cited stability of the RHDV genome across a broad
    geographic range and time as another reason not to be concerned about
    RHDV's potential for virulence, a narrow range of sequence variation is
    the expected result. The explosiveness and mortality of the RHDV syndrome
    imply a recent introduction of the virus into the species/genus. From
    where? Outbreak/epidemic strains tend to be more closely related to each
    other than strains collected after a pathogen has been in the host for
    many decades. Variation within human caliciviruses required application
    of broadly reactive methods, such as RT-PCR using conserved primers, to
    samples that were electon microscopy-negative. Have similarly
    comprehensive methods been applied to look for diversity of RHDV among
    field isolates?

    We know already that RHDV and the similar European Brown Hare Syndrome
    calivirus are different enough from each other to be considered
    potentially different serotypes, based upon the degree of difference
    observed between them.

    Although CSIRO has been focusing on the limited genetic variability of
    the rabbit calicivirus, apparently to provide reassurance as to the
    safety of the release, the existence of genomic and antigenic variability
    may be a "blessing" for the eradication program. It might be possible to
    adapt the Brown Hare rabbit calicivirus to the bunnies in Australia. If
    the genomic differences between the RHDV and EBHS caliciviruses correspond
    to antigenic differences, such adaption might permit a "two-hit" challenge
    to the wild Australian rabbits, permitting the eradication program to
    succeed. By focusing on the presumed "stability" and "limited variation"
    of RHDV, the CSIRO researchers may have "focused" themselves out of viable
    solutions that would make their rabbit calicivirus program really succeed.

4.  It is very difficult to get an understanding of CSIRO activities through
    in-house publications and press releases. Such documents are not readily
    available and do not engender the same peer review as formal publication.
    The fact that CSIRO devloped plans for the RHDV without publication in
    the scientific literature means that they do not appear to have availed 
    themselves of of peer comments that might have prevented the premature
    escape of the virus.

5.  Were experiments conducted by the CSIRO appropriate to evaluate the risk
    of interspecies spread? People I respect with experience in this area
    have doubts.

6.  What is the virulence factor that causes death in rabbits? Is this factor
    shared by other calicivirus strains?

7.  What is the specificity of the assays for RHDV and antibody to RHDV? What
    studies were done in humans and animals in Australia to document absence
    of cross-reacting antibodies to ensure that post-release surveillance
    would yield valid conclusions?

8.  What is the original source of the RHDV? Epidemiologic evidence of high
    mortality and rapid spread in rabbits suggest that RHDV was new to rabbits
    15 years ago or so. Where did it come from? Knowing where it came from
    would provide for good planning in a deliberate release.

Unfortunately, these concerns have not been given what I consider to be proper
attention by CSIRO or the supervisory board advocating this virus release. I
am hoping that a scientific forum can be developed where these concerns
are discussed by interested parties.


David O. Matson, M.D., Ph.D.
Center for Pediatric Research
Norfolk, Virginia