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ABSTRACT: An accurate diagnosis of infection by Leishmania infantum in dogs is fundamental for the control of zoonotic visceral leishmaniasis (VL). Histopathology (HP) and immunohistochemistry (IHC) are frequently used for the histological diagnosis of L. infantum in dogs, but have shown limited accuracy. To improve the sensitivity and specificity of the histological diagnosis of VL, we evaluated automated in situ hybridization (ISH) using a generic probe for Leishmania and a specific probe for L. infantum in surgical skin biopsies of dogs. The ISH results were compared with those of HP and IHC, using parasitological culture as the reference standard. Samples of skin from 51 dogs with cutaneous L. infantum infection and 51 non-infected dogs were randomly selected from samples of dogs from various cities in Brazil where canine VL is endemic. These samples were processed for parasitological culture, HP, IHC and ISH using both probes. The sensitivities of ISH using the specific probe, ISH using the generic probe, IHC and HP were, respectively, 74.5%, 70.6%, 69.5% and 57.6%. The specificity of both ISH probes tested was 100% and there was no cross-hybridization of the generic and specific probes with selected pathogenic fungi and protozoa. The specific probe discriminated L. infantum from the other species of Leishmania that infect dogs in the New World. ISH is highly sensitive and specific for the diagnosis of L. infantum in histologic samples of skin from infected dogs and can be used on routine biopsy material to make a diagnosis of leishmaniasis.
Journal of clinical microbiology 11/2012; · 4.16 Impact Factor
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ABSTRACT: A polyomavirus was isolated from the eyes of horses, and the sequence was determined. A nearly identical VP1 sequence was amplified from the kidney of another animal. We report the complete genome sequence of the first polyomavirus to be isolated from a horse. Analysis shows it to be most closely related overall to human and nonhuman primate polyomaviruses.
Journal of Virology 08/2012; 86(16):8903. · 5.40 Impact Factor
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ABSTRACT: Canid herpesvirus 1 (CaHV-1) is a well-known cause of fatal hepatic and renal necrosis in neonatal puppies. In adult dogs infected with CaHV-1, papulovesicular genital lesions may be observed. CaHV-1 infection during pregnancy can lead to embryonic resorption, abortion, and stillbirth. In high-density dog populations, CaHV-1 can also contribute to kennel cough. Furthermore, recent literature has clearly documented that CaHV-1 can induce ocular disease in immature and adult dogs. The current study describes a case of fatal CaHV-1 infection in a 9-year-old spayed female Bichon Frise dog. Following a history of vomiting and diarrhea, the dog deteriorated and subsequently died. The main lesions were multifocal areas of necrosis with intranuclear inclusion bodies in the liver, adrenal gland, and small intestine, similar to the lesions observed in CaHV-1-infected puppies. Infection with CaHV-1 was confirmed on samples of liver by polymerase chain reaction, immunohistochemistry, and in situ hybridization. There was no indication of immunosuppression in this dog. Based on the results presented herein, CaHV-1 should be included in the list of differential diagnoses of hepatic necrosis in adult dogs.
Journal of veterinary diagnostic investigation: official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 05/2012; 24(3):604-7. · 1.21 Impact Factor
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ABSTRACT: Immunohistochemistry (IHC) and in situ hybridization (ISH) can be used either to detect or to differentiate between Eastern equine encephalitis virus (EEEV) and West Nile virus (WNV) within formalin-fixed, paraffin-embedded (FFPE) brain tissue of horses. To compare the diagnostic sensitivity and specificity of ISH and IHC, FFPE brain tissue from 20 EEEV-positive horses and 16 WNV-positive horses were tested with both EEEV and WNV oligoprobes and EEEV- and WNV-specific antibodies. Reverse transcription polymerase chain reaction (RT-PCR) for detection of EEEV and WNV was used as the gold standard to confirm infection. All horses that tested positive for EEEV by RT-PCR also tested positive by IHC and ISH, except for 1 case that was false-negative by ISH. In contrast, all horses that tested positive for WNV by RT-PCR tested negative by IHC and only 2 horses tested positive by ISH. No false-positives were detected with either method for both viruses. Both IHC and ISH are highly specific and sensitive diagnostic methods to detect EEEV in equine FFPE brain tissues, although neither appear effective for the diagnosis of WNV in equine neurologic cases.
Journal of veterinary diagnostic investigation: official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 03/2012; 24(2):333-8. · 1.21 Impact Factor
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Sarah D Cramer,
Gregory A Campbell,
Bradley L Njaa,
Sandra E Morgan,
Stephen K Smith,
William R McLin,
Bruce W Brodersen, Annabel G Wise,
Gail Scherba,
Ingeborg M Langohr,
Roger K Maes
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ABSTRACT: Pseudorabies is caused by Suid herpesvirus 1, a member of the Alphaherpesvirinae subfamily. Although pigs are the natural host of Pseudorabies virus (PRV), the virus has a broad host range and may cause fatal encephalitis in many species. The United States obtained PRV-free status in 2004 after the virus was eradicated from domestic swineherds, but the virus is still present in feral swine populations. The current report describes PRV infection in 3 dogs that were used to hunt feral swine. The dogs developed clinical signs including facial pruritus with facial abrasions, dyspnea, vomiting, diarrhea, ataxia, muscle stiffness, and death. Two were euthanized, and 1 died within approximately 48 hr after onset of clinical signs. The salient histologic changes consisted of neutrophilic trigeminal ganglioneuritis with neuronophagia and equivocal intranuclear inclusion bodies. Pseudorabies virus was isolated from fresh tissues from 2 of the dogs, and immunohistochemistry detected the virus in the third dog. Virus sequencing and phylogeny, based upon available GenBank sequences, revealed that the virus was likely a field strain that was closely related to a cluster of PRV strains previously identified in Illinois. Though eradicated from domestic swine in the United States, PRV is present in populations of feral swine, and should therefore continue to be considered a possible cause of disease in dogs and other domestic animals with compatible clinical history and signs. Continued surveillance is necessary to prevent reintroduction of PRV into domestic swine.
Journal of veterinary diagnostic investigation: official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 09/2011; 23(5):915-23. · 1.21 Impact Factor
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ABSTRACT: Ferret systemic coronavirus (FRSCV) infection is associated with an emerging, highly fatal disease of ferrets. Enhanced macrophage tropism and the resulting induction of pyogranulomatous lesions are shared with feline infectious peritonitis virus (FIPV) infection in cats, but are not features of ferret enteric coronavirus (FRECV) infection. Comparative sequence analysis of the distal one-third of the genomes of one FRSCV and one FRECV strain showed that these two ferret coronaviruses share >96% nucleotide sequence identities in the membrane (M), nucleocapsid (N) and non-structural protein genes (partial polymerase, open reading frames [ORFs] 3 and 7b). The envelope (E) protein gene showed a moderate nucleotide sequence similarity of 91.6%. In contrast, nucleotide and amino acid sequence similarities observed with the spike (S) protein were only 79.5 and 79.6%, respectively. Twenty-one amino acid differences within a 195-199-amino acid C-terminal portion of the S protein were conserved between 3 strains each of FRSCV and FRECV. Both systemic and enteric strains were found to carry a single ORF 3 gene with truncated proteins observed in two out of three FRSCV strains examined. The two enteric strains analyzed each contained an intact ORF 3 gene. Phylogenetically, FRSCV is more closely related to FRECV than to other group 1 coronaviruses.
Virus Research 04/2010; 149(1):42-50. · 2.94 Impact Factor
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ABSTRACT: Infection with feline herpesvirus-1 (FHV-1) is a major cause of upper respiratory and ocular diseases in Felidae. We report the first complete genomic sequence of FHV-1, as well as the construction and characterization of a bacterial artificial chromosome (BAC) clone of FHV-1, which contains the entire FHV-1 genome and has the BAC vector inserted at the left end of U(L). Complete genomic sequences were derived from both the FHV-1 BAC clone and purified virion DNA. The FHV-1 genome is 135,797bp in size with an overall G+C content of 45%. A total of 78 open reading frames were predicted, encoding 74 distinct proteins. The gene arrangement is collinear with that of most sequenced varicelloviruses. The virus regenerated from the BAC was very similar to the parental C-27 strain in vitro in terms of plaque morphology and growth characteristics and highly virulent in cats in a preliminary in vivo study.
Virology 03/2010; 401(2):215-27. · 3.35 Impact Factor
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ABSTRACT: We report a disease outbreak in a Michigan rabbitry of a rabbit calicivirus distinct from the foreign animal disease agent, rabbit hemorrhagic disease virus (RHDV). The novel virus has been designated Michigan rabbit calicivirus (MRCV). Caliciviruses of the Lagovirus genus other than RHDV have not been described in US rabbit populations. The case-fatality rate was 32.5% (65/200). Clinical signs included hemorrhage and sudden death, with hepatic necrosis. Analysis of viral RNA sequence from >95% of the viral genome showed an average similarity of 79% with RHDV. Similarity of the predicted MRCV capsid amino acid sequence ranged from 89.8% to 91.3%, much lower than the 98% amino acid similarity between RHDV strains. Experimentally infected rabbits lacked clinical disease, but MRCV was detected in tissues by PCR. We propose that MRCV primarily causes subclinical infection but may induce overt RHD-like disease under certain field conditions.
Emerging Infectious Diseases 12/2009; 15(12):1955-62. · 6.79 Impact Factor
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ABSTRACT: OBJECTIVE- To determine clinical, histologic, and immunohistochemical findings for dogs with wart-like lesions involving the paw pads. DESIGN- Retrospective case series. ANIMALS- 24 dogs (18 Greyhounds and 6 dogs of other breeds). PROCEDURES- Medical records were reviewed for information on signalment, physical examination findings, concurrent disease processes, location of all lesions, and, when available, results of histologic examination of biopsy specimens. Available biopsy specimens (n = 11) were submitted for immunohistochemical staining and a PCR assay to identify viral inclusion bodies. RESULTS- In Greyhounds, most lesions involved the pads of the third and fourth digits, had a consistent histologic appearance without evidence of inflammation, were negative for papillomavirus, and had an unsatisfactory response to treatment. In other breeds, lesions often involved the pads of non-weight-bearing digits, had histologic evidence of inflammation, were positive for papillomavirus, and responded to surgical treatment. CONCLUSIONS AND CLINICAL RELEVANCE- Results suggested that wart-like lesions involving the paw pads of Greyhounds were a distinct clinical entity with features resembling porokeratosis plantaris discreta in humans. In Greyhounds, these lesions were not associated with an underlying viral etiology and, therefore, should not be considered plantar warts. Alternative treatments should be investigated because current treatments were generally unsuccessful in Greyhounds. Wart-like lesions of the paw pads in other breeds were often associated with papillomavirus, and surgical excision appeared curative.
Journal of the American Veterinary Medical Association 07/2009; 234(12):1555-8. · 1.79 Impact Factor
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ABSTRACT: Equine herpesvirus 9 was detected in a polar bear with progressive encephalitis; the source was traced to 2 members of a potential equid reservoir species, Grevy's zebras. The virus was also found in an aborted Persian onager. Thus, the natural host range is extended to 6 species in 3 mammalian orders.
Emerging Infectious Diseases 11/2008; 14(10):1616-9. · 6.79 Impact Factor
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ABSTRACT: A 5-month-old captive female striped skunk (Mephitis mephitis) was evaluated because of lethargy, signs of depression, azotemia, and erythema of the skin around the eyes.
Antemortem diagnostic tests revealed renal disease but failed to identify an etiologic agent. A diagnosis of severe nonsuppurative interstitial nephritis was made on the basis of results of histologic examination of renal biopsy specimens.
The skunk was administered isotonic fluids SC daily and later every other day because of the handling-related stress. Because of the skunk's deteriorating condition, it was euthanized after 24 days of supportive care. Aleutian disease was diagnosed on the basis of positive results of a PCR assay that targeted the DNA from Aleutian disease virus (ADV); positive results for ADV were also obtained by use of plasma counterimmunoelectrophoresis and an ELISA. Genetic sequencing of the 365-base pair PCR product revealed 90% sequence identity with mink ADV.
In the skunk of this report, infection with a skunk-specific parvovirus resulted in clinical signs and pathologic changes similar to those associated with ADV infection in mink. For skunks with signs of renal failure, differential diagnoses should include parvovirus infection. In confirmed cases of infection with this ADV-like virus, appropriate quarantine and biosecurity measures should be in place to prevent spread to other susceptible animals within a zoological collection.
Journal of the American Veterinary Medical Association 04/2008; 232(5):742-6. · 1.79 Impact Factor
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ABSTRACT: Diagnosis of canine parvovirus type 2 and feline panleukopenia virus infection in dogs and cats may be hampered by the severity of enteric lesions, secondary bacterial overgrowth, and rapid onset of autolysis. In contrast to small intestine, tongue epithelium is less sensitive to postmortem changes. Sections of tongue and small intestine from 11 dogs and 11 cats with a clinical history and gross and microscopic lesions compatible with canine and feline parvoviral infection were examined for parvoviral infection using real-time polymerase chain reaction (PCR), immunohistochemistry (IHC), and direct fluorescent antibody testing (FA). Parvoviral DNA was detected by PCR in both small intestine and tongue of all but 1 dog. Nineteen of 22 animals (86%) with suspect or positive FA staining in the small intestine also had positive FA and IHC staining in the tongue. Three of 3 dogs (100%) whose carcasses had been frozen and thawed prior to necropsy had more consistently positive staining in tongue than in small intestine by FA and IHC. These data confirm tongue as an excellent complementary sample for parvoviral testing in dogs and cats, especially in cases in which postmortem autolysis has occurred.
Journal of veterinary diagnostic investigation: official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 08/2007; 19(4):409-13. · 1.21 Impact Factor
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ABSTRACT: The objective of this study was to investigate whether intramuscular vaccination of healthy adult horses with a killed or a modified live equine herpesvirus type 1 (EHV-1) vaccine could induce transient positive PCR results in either blood or secretions collected on a nasopharyngeal swab. Four horses in each group received either a single killed or a modified-live vaccine intramuscularly. Two local commingled and 2 distant nonvaccinated controls were included for each group. All horses were observed daily for evidence of clinical abnormalities throughout the study periods. Blood and nasopharyngeal swabs were collected twice before vaccination and once weekly for 4 weeks after vaccination and submitted for PCR testing for EHV-1 by 2 independent laboratories using different real-time PCR methodologies. Serum samples collected from all horses on the vaccination day and 21 days later were tested for antibodies against EHV-1 using a serum neutralization test. Whereas the 2 vaccine strains tested positive in both EHV-1 PCR assays, nasopharyngeal swabs and whole blood collected from vaccinated and control horses had negative PCR test results for EHV-1 during the entire study period. Serum neutralization testing revealed a 2- to 4-fold increase in titers for all vaccinated horses, whereas titers in control horses were largely unchanged. The use of seropositive horses before immunization and the sampling frequency of 7 days may have prevented the occasional molecular detection of the vaccine virus in whole blood and nasopharyngeal secretions. However, the study results demonstrate that detection of EHV-1 DNA by PCR in vaccinated and unvaccinated healthy horses is not a common event.
Journal of veterinary diagnostic investigation: official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 06/2007; 19(3):290-3. · 1.21 Impact Factor
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ABSTRACT: Based on results of earlier studies, brain, heart and kidney are most commonly used for West Nile virus (WNV) detection in avian species. Both monoclonal and polyclonal antibodies have been used for the immunohistochemical diagnosis of WNV in these species. Thus far, no studies have been performed to compare the sensitivity and specificity of monoclonal and polyclonal antibodies in detecting WNV in American crows (Corvus brachyrhynchos). Our objectives were to determine 1) the comparative sensitivities of monoclonal and polyclonal antibodies for immunohistochemical (IHC) diagnosis of WNV infection in free-ranging American crows, 2) which organ(s) is/are most suitable for IHC-based diagnosis of WNV, and 3) how real-time RT-PCR on RNA extracted from formalin-fixed paraffin-embedded tissues compared to IHC for the diagnosis of WNV infection.
Various combinations, depending on tissue availability, of sections of heart, kidney, brain, liver, lung, spleen, and small intestine from 85 free-ranging American crows were stained using a rabbit-polyclonal anti-WNV antibody as well as a monoclonal antibody directed against an epitope on Domain III of the E protein of WNV. The staining intensity and the extent of staining were determined for each organ using both antibodies. Real-time RT-PCR on formalin-fixed paraffin-embedded tissues from all 85 crows was performed.
Forty-three crows were IHC-positive in at least one of the examined organs with the polyclonal antibody, and of these, only 31 were positive when IHC was performed with the monoclonal antibody. Real-time RT-PCR amplified WNV-specific sequences from tissue extracts of the same 43 crows that were IHC-positive using the polyclonal antibody. All other 42 crows tested negative for WNV with real-time PCR and IHC staining. Both antibodies had a test specificity of 100% when compared to PCR results. The test sensitivity of monoclonal antibody-based IHC staining was only 72%, compared to 100% when using the polyclonal antibody.
The most sensitive, readily identified, positively staining organs for IHC are the kidney, liver, lung, spleen, and small intestine. Real-time RT-PCR and IHC staining using a polyclonal antibody on sections of these tissues are highly sensitive diagnostic tests for the detection of WNV in formalin-fixed tissues of American crows.
BMC Infectious Diseases 02/2007; 7:49. · 3.12 Impact Factor
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ABSTRACT: The complete genomic DNA of a novel papillomavirus (PV) was isolated from a basosquamous carcinoma on the wing of an Egyptian fruit bat (Rousettus aegyptiacus). Initial short sequences of the E1 and L1 genes of this virus were retrieved by PCR with degenerate papillomavirus-specific primers, and the entire R. aegyptiacus papillomavirus type 1 (RaPV-1) DNA was then amplified by long template PCR, cloned and sequenced with a transposon insertion method. The RaPV-1 genome counts 7970 basepairs and contains the typical papillomavirus open reading frames (ORF) (E1, E2, E4, E6, E7, L1 and L2). Based on a concatenated alignment of the E1, E2, L1 and L2 open reading frames of RaPV-1 and 46 other human and animal papillomavirus type species, a neighbor-joining phylogenetic tree was constructed. This phylogenetic analysis shows that RaPV-1 has a close-to-root position in the papillomavirus evolutionary tree. Since RaPV-1 is only distantly related to other papillomaviruses (with maximally 50% nucleotide sequence identity across the L1 open reading frame), it cannot be assigned to one of the existing papillomavirus genera and therefore represents the first member of a novel, as yet unnamed, close-to-root papillomavirus genus. This is the first time a papillomavirus has been isolated and characterized from a member of the Chiroptera order.
Veterinary Microbiology 11/2006; 117(2-4):267-75. · 3.33 Impact Factor
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ABSTRACT: A 5-yr-old female Egyptian fruit bat (Rousettus aegyptiacus) had a small raised pigmented mass removed from the lateral canthus of the left eye. Six additional variably sized, raised, smooth to cauliflower-like skin masses were observed randomly distributed throughout the left wing membranes. Four masses were removed and diagnosed microscopically as basosquamous carcinomas and papillomas. Additional masses, removed 6 mo and 1 yr later, showed bony invasion and squamous differentiation. Immunohistochemistry detected positive intranuclear staining for bovine papillomavirus antibody in all samples. Polymerase chain reaction done on DNA extracts from formalin-fixed, paraffin-embedded tumor tissue amplified a 450 base-pair segment analogous to the L1 region of human papillomavirus types 96 and 5. Basic Local Alignment Search Tool analysis of sequenced amplicons suggests a novel chiropteran papillomavirus. To our knowledge, this is the first report of papillomavirus-associated carcinoma in a chiropteran species.
Journal of Zoo and Wildlife Medicine 07/2006; 37(2):193-6. · 0.38 Impact Factor
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ABSTRACT: A novel coronavirus, designated as ferret enteric coronavirus (FECV), was identified in feces of domestic ferrets clinically diagnosed with epizootic catarrhal enteritis (ECE). Initially, partial sequences of the polymerase, spike, membrane protein, and nucleocapsid genes were generated using coronavirus consensus PCR assays. Subsequently, the complete sequences of the nucleocapsid gene and the last two open reading frames at the 3' terminus of the FECV genome were obtained. Phylogenetic analyses based on predicted partial amino acid sequences of the polymerase, spike, and membrane proteins, and full sequence of the nucleocapsid protein showed that FECV is genetically most closely related to group 1 coronaviruses. FECV is more similar to feline coronavirus, porcine transmissible gastroenteritis virus, and canine coronavirus than to porcine epidemic diarrhea virus and human coronavirus 229E. Molecular data presented in this study provide the first genetic evidence for a new coronavirus associated with clinical cases of ECE.
Virology 06/2006; 349(1):164-74. · 3.35 Impact Factor
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ABSTRACT: To compare 5 methods of preparation of RNA from feline urine samples for use in a feline calicivirus (FCV), p30 gene-based, real-time reverse-transcriptase polymerase chain reaction (RT-PCR) assay.
Urine and blood samples from 6 specific-pathogen-free cats.
Aliquots of each urine sample (unmodified, centrifuged, or mixed with whole or hemolyzed blood) were spiked with FCV and serially diluted in urine. Serial dilutions of FCV in tissue culture medium were used as positive controls. Viral RNA was prepared via dilution and thermal inactivation (DT method), polyethylene glycol precipitation (PEG method), isolation with oligo(dT)25-coated magnetic beads (dTMB method), or extraction by use of 2 silica gel-based columns (RN or QA method). Lower detection limits and mean RT-PCR threshold cycle (Ct) values associated with each RNA preparation method and sample type were compared.
Because DT-prepared samples yielded negative results via RT-PCR assay, this method was not evaluated. Lower detection limits (TCID50/sample) for the assay in urine were 1950, 104, 11, and 7 for PEG-, dTMB-, RN-, and QA-prepared samples, respectively. For RN and QA preparations, Ct values were similar and significantly lower than those for dTMB and PEG preparations. Overall, urine modifications did not affect FCV RNA detection in dTMB-, QA-, and RN-prepared samples.
Of the methods evaluated, the RN and QA methods of RNA preparation were most appropriate for the FCV RT-PCR assay. An RT-PCR assay optimized for detection of FCV in feline urine may aid investigations of FCV-induced urinary tract diseases in cats.
American Journal of Veterinary Research 06/2005; 66(5):915-20. · 1.27 Impact Factor
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ABSTRACT: To evaluate 2 rapid, patient-side assays for detection of Cryptosporidium parvum in feces from neonatal calves with diarrhea.
Diagnostic test evaluation Sample Population-Fecal samples from 96 neonatal (1 to 30 days old) calves with diarrhea.
Results of the rapid assays were compared with results of microscopic examination of fecal smears that had been stained with diamant fuchsin stain.
One of the rapid assays correctly identified 56 of 62 (90%) fecal samples positive for C. parvum oocysts and 33 of 34 (97%) fecal samples negative for oocysts. The other assay correctly identified 53 of 62 (85%) fecal samples positive for oocysts and 33 of 34 (97%) fecal samples negative for oocysts.
Results suggest that these 2 rapid assays are accurate when used to detect C. parvum in fecal samples from neonatal calves with diarrhea.
Journal of the American Veterinary Medical Association 11/2004; 225(7):1090-2. · 1.79 Impact Factor
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ABSTRACT: Noroviruses have emerged as the leading worldwide cause of acute non-bacterial gastroenteritis in humans. The presence of noroviruses in diarrheic stool samples from calves on Michigan and Wisconsin dairy farms was investigated by RT-PCR. Norovirus-positive samples were found on all eight farms studied in Michigan and on 2 out of 14 farms in Wisconsin. Phylogenetic analyses of partial polymerase and capsid sequences, derived for a subset of these bovine noroviruses, showed that these strains formed a group which is genetically distinct from the human noroviruses, but more closely related to genogroup I than to genogroup II human noroviruses. Examination of 2 full and 10 additional partial capsid (ORF2) sequences of these bovine strains revealed the presence of two genetic subgroups or clusters of bovine noroviruses circulating on Michigan and Wisconsin farms. One subgroup is "Jena-like", the other "Newbury agent-2-like".
Virus Research 04/2004; 100(2):165-77. · 2.94 Impact Factor
