Paul W.

Reno and Katherine Klekis

Departrneilt oi !vf~crohraiogy d n d Mi gr,~tory Fish Rcbe'lrc h in\tlLute, Ilniversity of Marnc, Orono, A4E 04469, USA
Stuart W. Sherburne
St~lhe of Illaine. blepdrlmenr of Mdr,i-re Kcsourt er, i.V Uoothbay Harbor, (WE 04575, LISA
and Bruce L. Nieholssn
Department of hbtcrohrology and ~Wigratrrry Fish Researc 12 in\titutc, University of Maine, Orono, ,WE 04449, 113A
Reno, P. 1Y., I<. Kieftis, S. 1W. Sherburne, and 5. b. Ni chol ~on. 1986. Experimental infection and pathogenesis
of viral erythrocytic necrosis (VEN) in Atlantic cod, Cadus mor hu. Can. J . Fish. r2quat. lici. 4.3:
945-951.
ivlatu~c? Atlantic cod ( Zc~d~s rnorhuca) were experimentdl lv intected with viral erythrocytic necrosis (VEN) by
inoculation wi th washed erythrocytes ~4r cell-free homogendtes of erythrocytes from naturally infected fish.
Approximately one third of the animals exposed exhibited active infections. The temporal pattern of infection was
similar between naturally infected and experimental ty infected fish. One to two months alter infeceion, immdttrre
erythrocytes began to show clear evidence of VEN fol!owed by a rapid increase i n the proportion of infected
immature erythrocytes, frequently reaching 100%. A subsequent dramatic drtrp i n infection of immature erythro-
cytes occurred, coinciding with an increase of infection in mature erythrocytes. Significant crythrtzblastosis
occurred when the sverall erythrocyte infection rate reached ag~prowicnatcly TO%, but none of the newly
generated erythrocytes appeared infected. The peak infection rate (40-60% of erythrocytes infected) declined
slowly and the infection, i n must instcrnccs, ems completely resolved.
C>n a infect6 exp6rirnentalement des morues de IIAtlantrque (Cadus rnorhsrd) adultcs par la r16crose virale des
globules rouges (IdVCR) en leur inoculant des erythrocytes laves csu des brcsyats d'erythrocytes exempts de
cellules proversant de poissons contamin& ndturellement. En\ iron un tiers des poissons expos& ont rnontrb dss
signes d'infection. t e mode d'infection dar-is Ip temps &,lit scrnblalale pour le5 poissons contarnines expkri-
mentalerrpent et pour ceux qui l'ttaient naturellement. Entre un et deux mois ,lpr&s I'infection, les el-ythroc-ytea
irnrnatures ont csmn~enck A montrer des signes kvidents de NVGR et i l y a eu saebskquemrnent une augmentation
rapide de la proportion d16rythrocytes irnrnatures Infectks, c p i atteignatt souvent 100 %. Il y a eu par la suite une
baisse considtrable d'krytl~rocytes i n~m~l t ures i nf~ctes, qui coincidait avec une aalgrn~ntation du nornbre d'bry-
throcytes matures infect&. II y a eu une irnportantc 6rythrcsblastosc lorsquc le tdux global d'infection des
6rytfirocytss a atteint envirsn 10 %, mais aueua des brythrocytes nouvellcrnent forrnes n'a serntaliP contarnine.
Il y a eu une baisse lente du tatex maximal d'infection (40 i 60 %, des 6rythrocytes infect&) et l'infection, dans
la plupart des cas, s'est eompleternent r6sorbbe.
Received February 2 7 , 1985
;4ccepted l anudry 27, 1986
(J8826)
iral erythrocytic necrosis (VEN) has been identified
in a wide range of poikilothermic vertebrates including
fishes (Pappy et a!. 1976; Doyle 1971; Evelyn and
Traxler 1978; Johnston and Davics 1973; Laird and
Bullock 1969; Reno et al. 1978), amphibians (Bernard et al.
1968), and reptiles (Stehkns and Johnston 1966). The infec-
tion HS characterized by the presence of cytoplasmic inclusion
bodies visible by light micrc~scopy in infected erythrocytes.
Examraination by electron microscopy reveals that these
inclusions are associated with one of several types of large
(150-368 earn) icosohedral Qeoxyribovisuses pra~visionally
classified as members of the irido~~iricjae (Appy et al . 1976;
Evelyn and Traxler 1978; Wen<? et a], 1978; Srnail and
Egglestone t98O). Whereas all erythrocytic necrosis viruses
(ENV) associated with VEN in a of Pacific Ocean fish
Can. 9. Fish. Ayual. Sci., Vol. 43, 1986
species appear iclentisal in size and ultrastructure (Evelyn and
Traxler 1978; Machfillan and Muleahy 1979)- several appar-
ently species-specific ENV that differ markedly in size and
ultrastructure have been described in Atlantic Ocean fishes
(Appey et al. 1976; Johnston and Davies 1973; Reno et ale
19'98).
In vitro propagation of ENV has ahus far been unsuccessful
(Evelyn and Trawler 1948; Reno and Nicholson 1980). How-
ever, ENV of Pacific salme~nids (Onc.or&ayncus sp.) and herring
(Clidpeu harengus pcablcsi) has been successfaelty transmitted in
viva to juvenile salmon (Evelyn and Traxler 1978; hiacMi%Ean
and Mulcahy 1979: MacMillan et al. 1988). These studies
demonstrated that VEN infection in Pacific salmonids did not
cause mortalities directly but did result in severe anemia and
increased susceptibility to secondary bacterial infections and
945
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TIME kdayd
FIG. 1 . Time course of natural VEN i1.n adlala Atlantis cod. Mia1
infection rate 0.0 1 %. @, VEN-infected imlr-aature erythrocytes; A,
dotal evthrocydes infected; @, uninfected, immature erythrocytes,
adverse environmentah effects. It has recently been demon-
strated that VEN infection of Atlantic cod ( Gad~s B ~ M O Y ~ ~ U ~ ) and
herring (Cbup~a harengus harengeds) results in a moderate
worrnocytic anemia and alteration of erythrocyte r-kaetabolism
(Reno et al. 1985).
AEtt~ough ENV infection in most affected species has
been observed in evthrocytes in all stages of development, i t
is not known which cells are initially infected and little iwfor-
mation is available about the time course and pathogenesis
sf the infection.
The objective of this study was to determine if sod ENV
couid be transmitted to adult Atlantic cod and, if transmission
was accon~plished, to trace the development sf the infection
in comparison with that in naturally infected fish. This would
lead t s a better understanding of the disease process in cod
and help assess the potential effect sf the disease on wild
psp~glations.
Materials and Methods
For most studies on the experimental transmission sf ENV,
mature Atiantic cod, ranging in size from 30 to 50 crn total
length, were collected over a 2-yr per i d by drag net near
Boothbay Harbor, Maine. They we= held for 2-4 wk prior t s
use in 3-m-diameter circular plastic pools supplied with un-
treated, running seawater at ambient temperatures of 2-7OC.
For most infection experiments, fish were transferred to
2-m-diameter pools supplied with recirculating artificial sea-
water (Instant Ocean, Fisher Sci., Medford, MA) at tempera-
tures of ,$-- 10@.Fish were fed smelt sterilized by akatoc%aving.
For more detailed studies of the time course and pathogenesis
of disease, mature cod were eoBLected as before and held in
TABLE I . Experimental transfer of er-ydhrcacytic ncsrosis virus (ENV)
infection in cod.
Infection
rate of VEN
Exp. donor Preparation positive/wurnber
NCP . 6%) of i n ~ c ~ l u m' ~ exposed" (2 infection
Total
Washed E
Washed E
Wrbshed E
Washed E
E lysate
E lysatc
E lysaee
K lysate
"E = erythrocytes.
"ecipient fish were tested for VEN at least three times over a
period of several months.
qumntine for 2 wk in 3-rn plastic pools supplied with running,
untreated seawater. Afer the quarantine pel-iod. the fish were
housed in the same pools and used for infectivity studies.
Over 2 yr, several attempts were made to transmit ENV to
adult cod. Early studies were designed ts determine if the virus
could indeed be transmitted, while later attempts were made to
trace the developaa~ent of disease. Blsod was collected by
caudal vascuiar puncture from naturally infected cod as a
source sf virus for experimental transmission studies. Six
donor fish were used as a source of ENV during the course of
four attempts to transmit the virus to a total of over 68 fish.
Blood from apparently uninfected fish was similarly collected
for mock infection of 23 fish. Blood cells were washed three
times by centrifugation at 50 X g HW marine phosphate buf-
fered saline (MPBS) (Reno and Nicholson 1980) and the cell
pellet was resuspended in four volu~nes s f hfPBS. Fish to be
used for infectivity studies were held 2-4 wk before use and
then injected either intraperitoneaily (0.4 amL) or intravascu-
larly (0.2 mL) with washed whole blood cells or cell-free
erythrocyte lysates prepared by hypot~snic lysis.
Evidence for VEN infection was obtained by collecting
blood from the efferent branehial artery or caudal puncture9
preparing thin biood smears, staining with Giernsa, and exam-
ining by light microscopy as described previousty (Reno
et al. 1978). The erythrocyte infecticsn rate was expressed as
the percentage of VEN-infected erythrocytes per 588-600
erythrocytes.
For a more detailed study of the initiation, progression, and
pathogenesis of VEN, a single study was performed in which
14 cod were experlmentaliy or mock infected or not injected
and the blood sampled from each fish at weekly intervals for a
period of 6 ma to evaluate both the VEN status and the nuannbers
sf immature eqthrocytes in the peripheral eirculatisn. Since
there apparently are no recorded descriptions of the erythro-
poietic series in Atlantic cod, immature erythrocytes were clas-
sified sn the basis of their movhological simitarities to those
of other fishes (MacMillan and Mulcahy 1979; Sherbasrne
1973). Nonmyelogenoass ceBls which were mo~kologically
distinguishabIe from mature eqthrocytes, generally on the
basis sf cytoplasmic tint and shape, were considered to be
erythrocyte precursors.
Can. 9. Fish. Aqua. Sci ., V01. 43, 1986
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FIG. 2. Progress of experimental VEN infection in adult Atlantic cod. (A) E5 d after exposure. Immature erythrocyte (8) with characteristic
rounded shape and round nucleus exhibiting early effects of VEN: chromatin margination and coaEescencc and a small, round perinuclear
inclusion body (mow). (B) 50 d after exposure. Two immature erythrocytes showing VEN inclusions and severe karyorrhexis. No evidence of
VEN in mature erythrocytes. (C) 109 d after exposure. Approximately 40% of eke ~rythrocytes show evidence of severe nuclear degeneration
ehaacteristic of VEN. Marked eryehroblastosis has occurred, but none of the immature crythrocytcs (arrows) appear t c ~ be infected. Bar represents
10 pm.
Tissues from kidney, spleen, liver, heart, intestine, and
gonads were fixed in MPBS-buffered formalin (I@%), em-
bedded ir% paraffin, sectioned at 6 pm, and stained with hema-
tsxylin and eosin.
Results
Although during the course of these studies nearly 100 VEN-
infected Atlantic cod were obtained and followed through the
clinical phase of VEN on an individual basis, onEy infrequently
were fish found t s be in the initial phase of infection. The data
in Fig. 1 represent the course of infection of owe such animal;
approximately 20 others monitored during later phases of the
disease followed approximately the same temporal pattern.
When first examined, the erythrocytes of the fish depicted
in Pig. I had an infection rate of less than 8.81 %. This rate
gradually increased and peaked after 3 rns of captivity at
approximately 40% of the erythrocytes being necrotic; a
gradual decline in the erythrocyte infection rate thew ensued
until about half of the peak value was reached at the termination
of the experiment at 168 do Nearly 20 other Individu,~l s exarn-
ined during the recovery phase of the infection were able to
clear peripheral blood of necrotic erythrocytes after being held
for several months.
During the course of the infection, a marked erythroblastssis
occumed, commencing when the erythrocyte infection rate
reached between 5 and %e%%; erythrsblastosis peaked the same
time as VEN infection rate and declined at approximately the
same rate as erythrocyte infection. The shape of this curve was
similar to infection curves generated for other infected indi-
viduals, and generally, there was a comelation between VEN
infection rate and the proportion s f immature erythrocytes.
The most striking finding, however, was the course of infec-
tisn in immature erythrocytes. These were the first cells
Can. J . Fish. Aquar. Sci., Vol. 43, 1986
947
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RG. 3. Electron micrograph of eqythrcrcytes from expcrianeataEBy infected adult cc~cf 90 d after
exposure. Nunwrous hexagonal and pentagonal particles characteristic of EN!! are seen in the
cytoplasm. Bar rspresents 500 nm.
T.B~BLE 2. Development of VEN in individual cxperirnenta%ly iafcctcd cad.
Source of Days to Days to
blood for % ENV in Route of Outcome of appearance Maximurn infection rate maxixjln~urn level
Enoculum inocuIum inoculation" i nocul at i onhof $'EN (9; VEN positlve cells) of VEN
ENV+
ENV+
ENVS
EN!! S
ENV-"
ENV-"
ENV-"
ENV-*
E NV- ~
ENV-*
ENV-*
Not injected
Not in-jected
Not injected
",*pa = intraperitoneal; i. v. = intravenous.
' ~f t e r exposure, fish were examined weekly for 90- t 42 do
"Determined to be ENV-positive during quarantine, but ENV-negative at time of experiment.
"ock infection.
i n k cted and showed evidence of necrosis much more rapidly
and to a higher degree than did mature erythrocytes; in addi-
tion, the decline in infection rate s f inmature erythrocytes
was also precipitous, with the ma.jority of the infected imma-
ture cells occurring within the first month. Peak infection in
immature erythrocytes. in this case leBO%, was coincident with
erythroblastosis.
Similar patterns of infection have been observed in about 20
c~ther naturally infected cod sampleti sequentially. Quantitative
differences were noted primarily in the maximal infection rate,
which varied between 10 and 85%; the basic shape s f the
erythrocyte infection curve, however, varied little.
At no time during these experiments did any individuals
show any gross or histopathologid evidence of disease in
visceral organs including kidney. spleen, liver, heart, intestine,
and gonads.
In addition to ~nonitorinag the development of VEN in feral
cod, the ability to experameratally infect fish with ENV was
evaluated in order t s provide a system for ramre detaiIed inves-
tigations s f the pathogenesis of the infection. The results s f
experimental and nmck infection of several groups of cod over
a 2-yr period are shown in Table I . The success of experi-
948
Can. J. Fish. Aqual. Sci . , V01. 43, 6986
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DAYS
DAY S
FIG. 4. Time course of experienaental VEN infection in adult Atlantic cod (four individuals from two
exprimeraes). B, VEN-infected immature erythrocytes; @, total erythrc~cytss infected: 0, w i n -
fected, immature erythrocytes.
mental transmission varied araacsng the different experiments;
however, the number of infected fish showing evidence of
VEN by microscopic examination was 3 1 /63 (33%). Fish that
were mock infected by inoculating with erythrocytes from
VEN-negative donors did not develop VEN.
At the microscopic level, the appearance of ENV-infected
cells In experimentally infected cod was identical to that
observed in eqthrocytes of naturally inafected fish. Infected
erythrocytes showed evidence of severe nuclear degeweratic~n
including pyknosis, karyonhewis, and karyc~lysis and the char-
acteristic circular, eosinsphilic inclusion body, with satellite
punctifom bodies that are probably individual visions (Fig. 2).
Examination of infected erythrocytes by electron rnicrosccspy
revealed typical ENV: 380-358 nm diameter hexagonal and
pentagonal particles with a complex capsid and sore structure
(Fig. 3). By these criteria, the erythrocytes appeared to have
been infected with ENV.
A more extensive experiment was carried out in order to
identify the cell typegs) initially infected and follow the pro-
gression of ENV pathogenesis. Cod were experimentally in-
fected with ENV by both intravenous and irntraperitoneal
in=jectiun or mock infected and each was sampled at weekly
intervals to determine their VEN status. 'Fhe results of this
experiment are shown in Table 2. Three of four fish inoculated
with washed, infected eryrkrocy tes became VEN positive about
I mo postinoculation. A11 three had been exposed by intra-
peritoneal injection, while the fish that had been exposed by
intravenous injection into a caredal vessel remained MEN nega-
tive. A single fish inoculated intravascullarly with washed
erythrocytes from a VEN-negative donor became infected;
however, the rapid onset of VEN (7 d) indicated that this
individual probably was in the iracubation phase s f the disease
at the time of exposure.
As depicted in Fig. 4, the overall pattern of pathogenesis
of the experimentally induced infectious process followed that
of the natural infection; there was an initial heavy infection sf
insmature erythrocytes falloe%led by evidence of infection of
mature erythrocytes, and finakly, erythroblastosis consisting
Cara. J . Fish. Ayucmt. Sci., VoI. 43, 1986 949
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"6, VEN INFECTION
FIG. 5. Linear regression analysis of 96 VEN infection rate versus
% immature erythrocytes. N = naturally infected fish; A, B, and
C = experimentally infected fish. Equations for lines: N: r = 8.812,
Y = 8.76.~ + 3.26; A: r = 8.982, Y = 0 . 7 3 ~ + 6.1: B : r = 0.866,
U - 0 . 5 2 ~ + 4.1; C: 8- = 0.95, Y = 0.715.~ -t 3.40.
of VEN-negative immature erythrocytes. In both experi-
mentally infected and naturaBly infected fish, linear regression
analysis indicated a significant (B < 8.BE5) positive comelation
between the level of immature erythrocytes and percent of
infected erythrocytes (Fig. 5) . Scrrnc variability in the patterns
of infecticpn of individual fish was observed. For example,
aitho~agh the time interval during which immature erythrocytes
were seen to be infected was generally very short, one fish
showed evidence of infected Immature erythrc~cytes over virtu-
ally the entire sampling period (Fig. 4B). Also, whereas w-nost
fish progressed rapidly from a barely detectable erythrocyte
infectinn rate (<8.W1%(7/G) to the rnrixirnaam sate, one individual
(Fig. 4D) remained at a low level sf infection for more than
3 moo In contrast with the high leveas of immature erythrocytes
in infected fish, the proportion of these sells in the peripheral
b!o~d of over 100 VEN-negative fish examined microscopi-
caIIy was Bow (usually in the range of 5- 10% and never
exceeding, 15%)).
This study has demonstrated expel.imental tsasssmlssion sf
\'EN in Atlantic cod by inoculation sf either washed infected
e~tkrcacytes or a cell-free lysate of infected cells. SuccessfuS
transfer of disease sscuned In four of five experiments. There
was no esbvlsbss relationship between the intensity sf infection
in the donors and the success rate of transmission, although in
the experiment where infection was n~ot successfubal the donor
fish had the lowest infection rate (2%). The overall rate sf
successfuk &ansmission of VEK was 33%, 666-9096 lower than
that found in intraspecific transfer of VEN ina Oncorhyncus
(MacMillan and Mulchy 1979). This difference may be ac-
counted for by the differences iw the species used, and by the
differences in age of the recipient fish in the two studies.
MacMiBIan and Mealcahy (1973) found a strong relationship
between the size sf the recipient fish and both the time to first
evidence of infection ( 2 d for 0.3-g fish, 20 d for 25-g fish) and
for the intensity sf infection generated (60% for 8,3-g, 40% for
25-g chum salmon (0. keta)). Our experiments were limited
by necessity to adult fish (30-58 cm total length) which may
have reduced the success rate and intensities sf infection. In
addition, the viruses present in the erythrocytes of the two
species are movhslogicall?y quite distinct (350 nrn in cod,
145 nm in Oncorhyncees and C1&8g~ea) and may have variable
effects on the respective hosts.
The relatively Eow success rate of experimental transfer of
infection may have been due ts host defense mechanisms.
Since the fish used in these experiments were feral, no disease
histories were available for the individuals used and some may,
E W fact, have had VEN and recovered.Thus, some animals may
have been immune and therefore refractory to reinfection. Iw
this respect, it is interesting to note that erythrocytes from one
donor fish which had recovered from VEN failed to transmit
the disease to recipients (Table 2).
The large size of cod ENV, 350 nrn (Appy et al. 1976; Reno
and Nichstson l980; Walker and Sherburne 1974), precluded
filtration through bacteria-retaining filters as is usala!ly done
when attempting to determine viral etiojogy. However, the
unique rnsl-~pEakp%csgy and Iscation of the agent which was found
consistently with the presence of erythrscytic lesions indicate
that ENV was kmnsrnitked in these experiments.
The time course of VEN infection, both natural and experi-
mental, was protracted, with an extended incubation period
(approximately I mo) as well as a lengthy disease phase of
about 3 ms duration, Hn all instances in which the infection was
followed over a Bong period of time, complete res~ht i on
c~ccumed with WB) indication of infected erythrocytes remaining.
The disease, while chronic and nonlethal to fish held in cap-
tivity, has deleterious physiological consequences including
anemia (Reno et ai. 198559, which may seriously affect survival
in the wild.
The most important findi~sg was that the first cells to exhibit
infection were immature eqthrocytes, generally those in the
final stages sf rnaturaeisn. This is the first evidence that eryah-
rscyte precursors rather than the fully mature erythrocytes
themselves are the sites for initiation of ENV infection. The
course of infection appeared to begin in a population of irnrna-
tare erythrocytes and became evident as Intracytopkasmic incju-
sions and nuclear degradation approwi~nateBy 1 rno after expo-
sure. 1n .some instances, virtually ail immature erythrocytes in
the peripheral circulation were infected. There are no data in
the Iiterature that detail the rate of erythropoiesis in Atlantic
cod. ConsequentBy, it is difficult to assess the maturation rate
of erythrocytes in this species and, therefore, to more percisely
determine at what stage sf erythrocyte maturation ENV invades
the cell. Ht is likely, however, that the infection is established
at least 1 rno prior to visible lesions in the erythrocyte.
The eqthroblastosis which ceccuwed during the Iatter stages
sf the infection was probably elicited by the decline in the
funceiedna% capacity of ENV-infected erythrocytes. In order to
maintain adequate levels of oxygen in tissues, this adjustment
was probably necessary. Other studies in our labsratoq (Reno
et a!. k985) have indicated that VEN causes a moderate nos-
rnocytic anemia in Atlantic cod. This is in contrast with the
.severe anemia caused by VEN in Pacific saHmon (MacMillan
and MuIsahy 1949). We h8ve found no evidence of VEN in the
immature erjg..trocytes generated Eate in infection. Whether
the Hack of infection at this stage was due to specific immu-
Can. J . Fish. Atpat. Sci., V d . 43, 1986
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nological defenses or to insufficient duration of observation
remains conjectural. However, while maintaining cod obtained
during the declining VEN phase (often for more than 3 mo),
recrudescetmce of the disease has not sccuared (P. W. Reno,
unpubl . data).
Further work should be carried out on this disease to deter-
mine its impact on susceptible paspulatisws, its mode of traezs-
mission in the sea, and its potential far interspecies transfer as
well as in the basic biochemistry and immunology of the viral
agent. The chronic nature of this disease may lead to subtle
detrimental effects on cod pspuli~tions in csmparisora with
more lethal diseases but these effects nzay. wonetheEess, have a
severe impact on cod populations.
Acknowledgements
We acknowledge the invaluable assistance of the personnel of [kc
Maine Department of Marine Resources in rnaiintaining and sampling
some of the experimental fish. Funds were provided by grant
5-WO! -0HL.19 I64 from the National Herad, Lung, and Blood Institute
of the Kational institutes of Hcalth, grtant 7522347 from the
National Science Foundation, and by the Maine Agrictalture Experi-
ment Station. MAES Publication No. I 1 E 3.
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