Emerging Infectious Diseases
[Volume 6 No.1 / January - February 2000]

Research

Norwalk-Like Calicivirus Genes in Farm Animals

Wim H.M. van der Poel,* Jan Vinj,* Reina van der
Heide,* Maria-Inmaculada Herrera, Amparo Vivo, and
Marion P.G. Koopmans*
*National Institute of Public Health and the
Environment, Bilthoven, the Netherlands; and Instituto
de Salud Carlos III, Majadahonda, Madrid, Spain

--------------------------------------------------------

 Viruses closely related to Norwalk-like
 viruses (NLVs) were recently found in
 stored stool samples from two calves
 (United Kingdom and Germany) and four pigs
 (Japan), sparking discussions about the
 potential for zoonotic transmission. To
 investigate if NLVs are commonly present in
 farm animals, pooled stool samples from 100
 pig farms, 48 chicken farms, 43 dairy cow
 herds, and 75 veal calf farms from the
 Netherlands were assayed by reverse
 transcription-polymerase chain reaction
 amplification, using primers specific for
 the detection of NLVs from humans. NLV RNA
 was detected in 33 (44%) of the specimens
 from veal calf farms and two (2%) specimens
 from pig farms. Our data show that NLV
 infectionsuntil recently thought to be
 restricted to humansoccur often in calves
 and sometimes in pigs. While zoonotic
 transmission has not been proven, these
 findings suggest that calves and pigs may
 be reservoir hosts of NLVs.

Caliciviruses infect animals and humans (1). Within the
family Caliciviridae, four genera have been
distinguished: vesivirus, lagovirus, Norwalk-like
viruses (NLV), and Sapporo-like viruses (SLV) (2). The
genera vesivirus and lagovirus contain a broad range of
animal caliciviruses, but viruses in the NLV and SLV
genera until recently have been found only in humans.
In recent years NLVs, also known as small
round-structured viruses, have emerged as a common
cause of infectious gastroenteritis in all age groups
and the main cause of outbreaks of gastroenteritis in
restaurants and institutions such as nursing homes and
hospitals (3-5).

Genetically, the human caliciviruses separate into at
least three genetic clusters, called genogroups (GG),
i.e., GGI and GGII NLV and the GGIII SLV (6). Each of
these genogroups comprises genomically and
antigenically diverse strains (7,8). This high degree
of diversity results in at least 13 distinct genotypes
for the NLVs (3,5,8-10). Many types of NLV cocirculate
in the general population, causing sporadic cases and
outbreaks. However, occasionally epidemics occur in
which most outbreaks are caused by a single genotype
(e.g., Lordsdale-like virus in the Netherlands in 1996)
(5,11). Hypotheses for the mechanisms behind the
emergence of epidemic types range from large-scale
foodborne transmission of a single strain to
introduction from a nonhuman reservoir. An indication
for the latter was a recent report from Japan of the
finding of NLV-like sequences in stool specimens from
pigs (12). Enteric caliciviruses have also been
detected in other animal species (calves, dogs, and
chickens) (13-16). However, except for canine
calicivirus (17), these viruses lack definitive
sequence evidence linking them to the Caliciviridae
(18). Molecular characterization of three calicivirus
strains from cattle has recently been reported. The
first virus (Tillamook virus; BCV-Bos1), described by
Neill et al. (19), caused respiratory symptoms and was
phylogenetically related to San Miguel sea lion virus
and vesicular exanthema of swine virus, both within the
genus vesivirus. Viruses in this genus have a broader
host range (20). Two other viruses closely related to
GGI NLVs (Jena virus 117/80 and Newbury agent type 2)
were detected in feces from newborn calves with
diarrhea (21,22). The similarity of the porcine and
bovine sequences with those of NLVs found in humans
suggests that a reservoir for these human pathogens may
exist in farm animals.

We studied enteric caliciviruses in recently collected
fecal samples from cattle, chicken, and swine in the
Netherlands by using polymerase chain reaction (PCR)
assays specific for NLVs found in humans. In addition,
we analyzed (by sequence analysis) the genetic
variation of detected calicivirus strains and compared
it with that of prototype GGI and GGII NLV strains.

Materials and Methods

Fecal Specimens

Stool specimens from animals were collected at farms in
the Netherlands as part of ongoing surveillance for
potential zoonotic microorganisms associated with
gastroenteritis in humans. From October 10, 1998, to
April 21, 1999, samples were collected from 3- to
9-month-old fattening pigs at 100 pig farms of 22 to
1600 pigs. From January 1 to April 1, 1998, stool
samples were collected from 75 veal calf farms of 38 to
930 calves and 43 dairy cattle herds of 25 to 180 cows.
The age of the calves was 1 to 52 weeks (average 12
weeks), and of dairy cattle herds 4 to 6 years. From
January 6 to April 21, 1998, samples were collected
from < 6-week-old chickens at 48 broiler farms, with
5,000 to 70,000 per farm.

Sampling

The sampling strategy allowed monitoring for pathogens
in a large number of animals and detection of
microorganisms at farm level with a prevalence of 5%
and 95% confidence (23). Fecal samples from calves,
pigs, and chickens (20 to 60 per farm) were collected
from animals housed in one randomly chosen farm
building. Dairy cattle of one farm were regarded as a
single herd, and stool samples were collected from
randomly selected cows. Until tested, fecal samples
were stored at -70C in 15 g/L of Trypton Soya broth
(TSB) (Oxoid CM 129) and 10% glycerol.

Molecular Detection of NLVs by RT-PCR

For extraction of viral RNA, stool samples were resuspended in HBBS Hanks (Gibco BRL, Breda, Netherlands) to a final concentration of approximately 10%. These suspensions were centrifuged at 3,000 x g for 20 minutes, and 100 l was used for RNA extraction by addition of a high-molarity solution of guanidinium isothiocyanate (GuSCN). Bound RNA was washed and eluted as previously described (24).

To reduce the risk for contamination, specimens from different species were analyzed separately, and one negative control sample was included for every two stool specimens. A human stool sample positive for NLV by electron microscopy was included as positive control. 

We used a single-round reverse transcription (RT)-PCR assay with a broadly reactive primer pair, which had been developed for the detection of NLVs in stool specimens from humans (5). For RT, 5 l RNA was mixed with 4 l 50 pmol JV13 primer. The solution was heated to 94C for 2 minutes, cooled, and 6 l RT buffer was added. The RT reaction was performed in a final volume of 15 l, consisting of 10
mM Tris-HCl (pH 8.3), 50 mM KCl, 3 mM MgCl2, 1 mM each of
deoxynucleoside triphosphates (dNTPs), and 5 units of avian
myeloblastoma virus RT (Boehringer Mannheim, Almere, Netherlands). The mixture was incubated for 1 hour at 42C, heated for 5 minutes at 94C to denature the enzyme, and then cooled. Five l of the RT mixture was added to the PCR mix, containing 10 mM Tris-HCl (pH 9.2), 75 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTPs, 2.5 units AmpliTaq (Perkin Elmer, Nieuwerkerk a/d IJssel, Netherlands), and 15 pmol primer JV12. Mineral oil was added, and 40 amplification cycles, 1 minute at 94C, 1.5 minute at 37C, and 1 minute at 74C each, were performed. The amplification products were analyzed by 2% agarose gel electrophoresis and visualized with UV after ethidium bromide staining.

For Southern blots, the RT-PCR products in the agarose gel were denatured by incubating in 0.5 M NaOH for 30 minutes and transferred to a positively charged nylon membrane (Boehringer, Almere, Netherlands) by vacuum blotting (Millipore, Etten-Leur, Netherlands). Hybridization of NLV RT-PCR products was performed as described previously (5).

Sequence and Phylogenetic Analysis

The NLV RT-PCR products of expected size (326 bp) and 21 products of smaller size of the calf herd samples were excised from a 2% agarose gel and purified with a Qiaquick gel extraction kit (Qiagen, Hilden, Germany). Purified RT-PCR products were sequenced with the BigDye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer Applied
Biosystems, Foster City, CA) by using PCR primers. Nucleotide sequences were edited by using SeqEd (V1.03, Applied Biosystems), aligned by Geneworks (V2.5, Intelligenetics, Mountain View, CA), and imported into the Treecon software package (25). The confidence
values of the internal nodes were calculated by performing 100 bootstrap analyses. Evolutionary trees for nucleotide sequences were drawn by using the Neighbor-joining method.

Electron Microscopy

Electron microscopy was performed as recommended by Flewett (26) and Doane and Anderson (27), with model Philips 400T (Philips, Eindhoven, Netherlands) at 80 kV. Identification of virus particles was based on morphologic criteria, i.e., the size and characteristic surface morphology (28).

Figure 1. Results of ethidium bromide staining (Panel A) and corresponding Southern hybridization (panels B and C) of
RT-PCR products of eight calf herd (CH) samples....
(Figures not available in ascii)

Results

Twenty-five (33%) of 75 veal calf farm samples 
and 2 (2.0%) of 100 pig farm samples showed
visible products of the expected size (326 bp).
The gel electrophoresis and hybridization
results are shown for eight strains (Figure 1).
Hybridization with a set of probes used for 
detection of NLVs in humans yielded no positive
reactions (Figure 1B); therefore, 46 products 
(25 of the expected size and 21 of a smaller
size [Figure 1A, lane 7]) were sequenced. All 25 
RT-PCR products of the expected size containedthe GLPSG amino acid motif characteristic of 
viral RNA polymerases (29). The 21 products of
smaller size revealed no viral sequences. The
NLV-RT-PCR was negative for all pooled specimens
from 43 dairy herds and 48 chicken farms.
By phylogenetic analysis, all calf calicivirus
sequences formed a tight cluster closely related
to NLVs from GGI with the highest similarity
with the Newbury calf calicivirus (Figure 2)
(22). Nucleotide sequence identities between the
calf NLVs and GGI NLVs were 63% to 70% (75% to
77% amino acids). The swine virus sequences
clustered as a separate lineage within GGII
NLVs, with 69% to 71% nucleotide and 79% to 83%
amino acid identity. In addition, the swine
sequences strongly resemble (94% nucleotides, 
100% amino acids) the swine calicivirus 
sequences from Japan. Partial polymerase 
sequences of Bo/NLV/176/1998/NET and

Figure 2. Phylogenetic relationships among human and
animal NLVs clustering in genogroup (GG) I and II, based on a 145-bp nucleotide sequence withing RNA polymerase gene....
(Figures not available in ascii)

Sw/NLV/34/1998/NET have been assigned GenBank Phylogenetic
accession numbers AF194183 and AF194184, relationships
respectively.  Since the calf and pig strains did not hybridize to the probe mixture for detection of NLVs from    
humans, specific probes were developed based on 
the consensus sequence of RT-PCR products of 10 
calf herd samples and on the pig farm samples,
respectively. The probe sequences were:  
RH1(calf) (5'-GGATGTGGTGCAGGCAA AC-3') and
RH2(pig) (5'-TCCGCATCTCTATCGT GG-3').
Hybridizations with the calf probe (RH1)
confirmed all RT-PCR-positive calf samples after
15 minutes exposure to an ECL hyperfilm (Figure
1C). Overnight exposure to ECL hyperfilm
revealed eight more positives, for 33 (44%)
positive calf farm samples.

Figure 3. Electron microscopy showing NLV particles in
a calf herd sample (CH176), negatively stained
with 2% K-PTA, pH 7.0. Bar - 40 nm.
(Figures not available in ascii)

All veal calf farm samples were screened by
electron microscopy for viruses (Table).
Particles with NLV morphologic features were
found in only one specimen (Figure 3), in which
the presence of calicivirus sequence was
confirmed by RT-PCR and hybridization (Table).

Conclusions

Until recently, humans were considered the sole 
host of NLVs. Recent studies in Japan and the
United Kingdom, however, demonstrated
calicivirus sequences in the caecum of pigs and
in stored calf stool samples containing
calicivirus-like particles by electron
microscopy (12,21,22). Molecular characterization of calf enteric caliciviruses, named Newbury agent (22) and Jena virus (21), revealed that they were genetically related
and more closely associated with GGI NLVs than with any other known calicivirus. In this study, we detected NLV nucleotide sequences in 33 (44%) of 75 of the pooled samples from veal calf farms.

Phylogenetic analysis of the detected sequences showed that they formed a tight cluster with the Newbury sequence (22) and were closely related to NLVs from GGI. Bridger et al. (30) demonstrated that calves can be infected with Newbury calicivirus. The detection of Newbury-like NLVs in pooled veal calf samples of 33 farms from different regions suggests that the species is a normal NLV host.
Similarly, the close genetic relationship between calicivirus sequences detected in the pig samples in our study and those from Japan suggests that these viruses commonly infect pigs. The close similarity of porcine and bovine sequences to the NLVs infecting humans indicates the possibility of an animal reservoir for human infection. However, these findings by no means prove zoonotic
transmission. Genetically related viruses are commonly found in different species without resulting in apparent widespread interspecies transmission (e.g., rotavirus). On the other hand, zoonotic transmission cannot be excluded. The genetic distances between the animal and human NLVs are similar to the distances between the GGI and GGII strains, and epidemic spread of some NLV strains (5) led to the widespreadpossibly globalemergence of a single
predominant strain in the human population in 1995-96 (11). This epidemiologic observation resembles early descriptions of the vesicular exanthema of swine virus (VESV) epidemics (31). These viruses, belonging to another genus within the Caliciviridae, the vesiviruses, are known for their broad host range (20). The occasional epidemic spread of VESV was later linked to introduction of caliciviruses from an ocean reservoir (20). Therefore, interspecies transmission of NLVs is possible; the occasional widespread NLV epidemics caused by a single strain may result from introductions of new NLV strains from an animal reservoir. 

Table. Viruses and virus particles detected by electron
 microscopy and corresponding Norwalk-like virus (NLV)
 RT-PCR results (including hybridization with a calf-NLV
 specific probe) in 16 of the 75 calf herd samples
(Table not available in ascii)

Reynolds et al. reported electron microscopy detection of
caliciviruses in calf diarrhea outbreaks (32), and the bovine caliciviruses Newbury agent (22) and Jena virus (21) were pathogenic for calves under experimental conditions and in field studies (33). In our study, we did not record disease or death on the surveyed farms and therefore cannot determine whether these caliciviruses were associated with disease. Despite repeated attempts, experimental infection of different animal species with the prototype Norwalk
virus has not been successful, with the exception of infection in chimpanzees (3). Our findings may lead to the development of an animal model for the NLVs of humans; for example, a pig model would enable studies of (mucosal) immunity following NLV infection (1).

The low prevalence of Norwalk-like calicivirus in swine farms (2 per 100) is in agreement with the findings reported from Japan (12). Electron microscopy confirmed the calicivirus origin of the detected NLV sequences in only one calf sample. This low number of positives by electron microscopy was not surprising, given the greater sensitivity of RT-PCR and the use of pooled specimens for screening.
NLVs in humans typically are shed at relatively low levels.

The absence of NLV sequences in all 43 dairy herd specimens may be explained by use of an inappropriate primer pair, which was optimized for detection of NLVs of humans (4) or by an acquired strain-specific immunity in older animals that prevents repeated infections. A third possibility is that levels of shedding in adult animals are very low,
precluding virus detection in pooled specimens. The swine farm samples were all from fattening pigs at least 3 months old. We may have found a higher prevalence in recently weaned pigs, as the prevalence of other enteric pathogens is highest in young piglets (34).

Our findings raise important questions about the host range of NLVs. It is unclear if animal NLVs form genetically distinct stable lineages or are part of a common pool of viruses circulating between animals and humans. If calves or swine indeed are reservoirs, the prevalence of calicivirus should be determined in both healthy and diseased cattle and swine and methods to detect such interspecies transmissions at an early stage should be developed, in collaborative
research by public health and veterinary sciences.

Acknowledgments

We thank A.W. van de Giessen and ing. W.D.C. Deisz for volunteering fecal specimens from the monitoring study for zoonotic enteric pathogens, and A.M. Henken and A.M. de Roda Husman for critically reading the manuscript.

This research was financially supported and approved by the Dutch Inspectorate for Health Protection, Commodities and Veterinary Public Health.

Dr. van der Poel is a veterinary virologist in the Microbiological Laboratory for Health Protection, National Institute of Public Health and the Environment, the Netherlands. His research involves viral zoonoses and foodborne virus infections.

Address for correspondence: Wim H.M. van der Poel, Microbiological Laboratory for Health Protection, National Institute of Public Health and the Environment, Antonie van Leeuwenhoeklaan 9, 3720 BA Bilthoven, the Netherlands; fax: 31-30-274-4434; e-mail: Wim.van.der.Poel@rivm.nl.

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