Transatlantic spread of highly pathogenic avian influenza H5N1 by wild birds from Europe to North America in 2021.

Highly pathogenic avian influenza (HPAI) viruses of the A/Goose/Guangdong/1/1996 lineage (GsGd), which threaten the health of poultry, wildlife and humans, are spreading across Asia, Europe, Africa and North America but are currently absent from South America and Oceania. In December 2021, H5N1 HPAI viruses were detected in poultry and a free-living gull in St. John's, Newfoundland and Labrador, Canada. Our phylogenetic analysis showed that these viruses were most closely related to HPAI GsGd viruses circulating in northwestern Europe in spring 2021. Our analysis of wild bird migration suggested that these viruses may have been carried across the Atlantic via Iceland, Greenland/Arctic or pelagic routes. The here documented incursion of HPAI GsGd viruses into North America raises concern for further virus spread across the Americas by wild bird migration.

Complete sequencing of Tunisian redspotted grouper nervous necrosis virus betanodavirus capsid gene and RNA-dependent RNA polymerase gene.

Finfish nodaviruses (betanodaviruses) can cause highly destructive infections in numerous species of farmed marine fish larvae and juveniles worldwide. The betanodavirus genome consists of two single-stranded positive-sense RNA molecules (RNA1 and RNA2). The virus can be classified into four genotypes based on the partial sequences of the coat protein (CP) gene (T2 and T4 regions). Currently, genomic sequence information for RNA1 regions of RNA2 outside of T2 and T4 is less well documented. This study reports on the characterization of the full RNA2 sequence of a Tunisian betanodavirus with a length of 1433 nt, containing a 339 amino acid open-reading frame encoding the CP, and typing to the redspotted grouper nervous necrosis virus Ia genotype following phylogenetic analysis. The homology of the capsid protein to other betanodaviruses or alphanodaviruses was compared. In addition, a full length RNA1 sequence of 3104 nt encoding a 982 amino acid RNA-dependent RNA polymerase was obtained.

Porcine circovirus 2 inclusion bodies in pulmonary and renal epithelial cells.

Porcine circovirus 2 (PCV2) is the cause of postweaning multisystemic wasting syndrome (PMWS). The most common lesions of PMWS are lymphohistiocytic to granulomatous lymphadenitis, interstitial pneumonia and interstitial nephritis, with intracytoplasmic amphophilic botryoid inclusion bodies in macrophages. In addition to these typical changes, intracytoplasmic botryoid inclusion bodies were observed in bronchial, bronchial glandular, and renal tubular epithelium of several pigs from 4 different farms in Western and Eastern Canada. PCV2 inclusion bodies were demonstrated to be located in the cytoplasm of epithelial cells by immunohistochemical staining for PCV2 and cytokeratin antigens and by ultrastructural demonstration of viral particles in the inclusion bodies within renal tubular epithelium.

Mortality event in freshwater drum Aplodinotus grunniens from Lake Ontario, Canada, associated with viral haemorrhagic septicemia virus, type IV.

A mortality event primarily affecting freshwater drum Aplodinotus grunniens was noted during April and May 2005 in the Bay of Quinte, Lake Ontario, Canada. A conservative estimate of the number of dead drum was approximately 100 metric tonnes. Large numbers of dead round goby Neogobius melanostomus were also seen, as well as a few muskellunge Esox masquinongy. In the drum, there was a consistent histological pattern of variably severe panvasculitis, a necrotising myocarditis, meningoencephalitis and a segmental enteritis. Moderate numbers of bullet-shaped viral particles consistent with a rhabdovirus were identified by transmission electron microscopy (TEM) in affected heart tissue. Following primary isolation from pooled tissues on fathead minnow (FHM) cells, a morphologically similar virus, approximately 165 x 60 nm in size, was visualised. Identification of the isolate as viral haemorrhagic septicemia virus (VHSV) was confirmed by enzyme immunoassay and by polymerase chain reaction. An appropriately sized product (468 bp) of the G-glycoprotein gene (nucleotides [nt] 340 to 807) was generated with RNA extracted from FHM cell supernatant. Analysis of a 360 nt partial glycoprotein gene sequence (nt 360 to 720) indicated a 96.4 to 97.2% nucleotide identity with known strains of North American (NA) VHSV. Analysis using Neighbour-joining distance methods assigned the isolate to the same lineage as the NA and Japanese isolates (Genogroup IV). However, there was sufficient sequence divergence from known NA VHSV isolates to suggest that this isolate may represent a distinct subgroup. The effects of ongoing mortality in freshwater drum and in multiple species during spring 2006 suggest that this newly recognised virus in the Great Lakes will have continued impact in the near future.

Estimation of the repeatability and reproducibility of three diagnostic tests for infectious salmon anaemia virus.

Reverse transcriptase polymerase chain reaction (RT-PCR), virus isolation (VI) and indirect fluorescent antibody tests (IFAT) are three assays currently used by the salmon industry to identify fish infected with infectious salmon anaemia virus (ISAV). However, no data are available on the repeatability (within-laboratory consistency) and reproducibility (between-laboratory consistency) of these assays and very limited information is available on the effect of freezing samples on test results. In order to evaluate these assays, five laboratories participated in a blinded study of 400 kidney samples representing four populations of farmed Atlantic salmon with different prevalence of ISAV. Each laboratory used its own testing protocols. Repeatability and reproducibility were evaluated using kappa as the measure of agreement. The effect of freezing was evaluated using the McNemar test. Freezing did not affect VI but improved the sensitivity of RT-PCR. The repeatability and reproducibility of VI was almost perfect. There was a substantial difference in repeatability of RT-PCR among the three laboratories with kappa ranging from 0.5 to 0.96. The repeatability for RT-PCR was generally low. The repeatability of IFAT was moderate when the results were analysed using 1+ and above as a positive result. The results of the study show the need to standardize the protocol and interpretation of RT-PCR.

Prevalence of hepatitis E virus antibodies in Canadian swine herds and identification of a novel variant of swine hepatitis E virus.

Swine hepatitis E virus is a newly identified potentially zoonotic virus from pigs of particular concern for possible direct transmission to a human xenotransplant recipient by organ transplantation. In the present study, prevalence of serum antibodies to hepatitis E virus was examined in Canadian swine herds. A total of 998 serum samples collected from 6-month-old healthy slaughter hogs were examined by enzyme immunoassay and Western blot analysis for antibodies to the recombinant open reading frame 3 (ORF3) protein of hepatitis E virus expressed in Escherichia coli. These samples represented more than 80 different swine production units from five major swine-producing provinces across Canada. From this study, 594 samples (59.4%) were found to be positive for hepatitis E virus antibody. The seroprevalence was higher in Quebec (88.8%) and Ontario (80.1%) than in Alberta and Saskatchewan (38.3%). By PCR using a pair of oligonucleotide primers deduced from the ORF2 sequence of human hepatitis E virus, a specific hepatitis E virus sequence was recovered from feces of pigs. The nucleotide sequence identity between the U.S. swine hepatitis E virus and the Canadian isolate (SK3) was only 85.8%, suggesting that genotypic variations may exist in swine hepatitis E virus in North America. Among 165 serum samples collected from humans in Saskatchewan, 2.4% were found to be positive for antibodies to the hepatitis E virus ORF3 protein. Our data indicate that hepatitis E virus is highly prevalent in commercial swine populations in Canada and support the suggestion that the swine hepatitis E virus may be an important zoonotic agent for humans.

Detection of bovine herpesvirus 1 in the semen of experimentally infected bulls by dot-blot hybridisation, polymerase chain reaction and virus isolation.

Two 18-month-old bovine herpesvirus 1 (BHV1)-seronegative bulls were inoculated experimentally with BHV1 via their prepuces. Semen collected at intervals was examined by optimised virus isolation, dot-blot hybridisation and the polymerase chain reaction (PCR) for detection of BHV1, and the infection was monitored serologically by using a virus neutralisation test. Antibodies were first detected 10 days after inoculation and were still present 40 days after inoculation. Semen collected from four to 40 days after inoculation was positive by PCR with Southern blot hybridisation whereas only the semen collected on day 4 was positive by dot-blot hybridisation, virus isolation and PCR with ethidium bromide staining. These results indicate that the bulls started to shed the virus in semen before they developed any detectable antibody. PCR with Southern blot hybridisation was the most sensitive of the three methods and detected virus for the longest period.

Comparison of dot blot hybridization, polymerase chain reaction, and virus isolation for detection of bovine herpesvirus-1 (BHV-1) in artificially infected bovine semen.

Bovine semen samples spiked with bovine herpesvirus 1 (BHV-1) were used to compare dot blot hybridization, polymerase chain reaction (PCR), and virus isolation for detection of BHV-1 in bovine semen. The PCR amplification used primers targeting the BHV-1 thymidine kinase gene and a nucleic acid releasing cocktail (GeneReleaser); the PCR product was used as the DNA probe in dot blot hybridization; virus isolation was done in primary bovine fetal testis (BFT) cell cultures. Semen diluted 1:20 in tissue culture medium had the least cytotoxicity and inhibition of viral cytopathic effects in BFT cells, allowing detection of 1 TCID50/100 microL of BHV-1 suspension by virus isolation. The presence of foreign DNA such as bovine sperm DNA or salmon sperm DNA increased the sensitivity of dot blot hybridization in detecting BHV-1, allowing detection of 20,000 TCID50/100 microL of neat semen. The inhibition of PCR amplification of BHV-1 DNA in bovine semen was eliminated by diluting the samples 1:20 in tissue culture medium. The best PCR amplification was obtained when semen was diluted 1:20 and when a reaction buffer of pH 9.0, with 1.0 mM MgCl2 was used. Under these conditions, the PCR followed by ethidium bromide staining of agarose gels could detect 1 TCID20/100 microL of sample, whereas PCR followed by Southern blot hybridization could detect 0.01 TCID50/100 microL of sample.(ABSTRACT TRUNCATED AT 250 WORDS)

Establishment of conditions for the detection of bovine herpesvirus-1 by polymerase chain reaction using primers in the thymidine kinase region.

Polymerase chain reaction (PCR) for detection of bovine herpesvirus-1 (BHV-1) was developed and optimized using 22 bp sense and 20 bp antisense primers in the thymidine kinase (TK) coding region. The amplification product is 183 bp long. The PCR optimization was done using BHV-1 tissue culture supernate (BHV-1TCS), concentrated BHV-1 tissue culture supernate (cBHV-1TCS) and sucrose gradient purified BHV-1 (pBHV-1). The sensitivity of four methods of sample preparation which are standard DNA extraction, modified proteinase K (PK) digestion, GeneReleaserTM + 34 cycles or + 44 cycles, and boiling were compared with virus isolation (VI) using BHV-1TCS. The incorporation of 10% glycerol in the reaction mixture, the incubation in PK for 18 hours and predenaturation of samples and cooling in ice prior to PCR were essential for the amplification of BHV-1 DNA for samples prepared by standard DNA extraction and modified PK digestion. The preparation of samples by Gene-ReleaserTM, a proprietary nucleic acid releasing cocktail, showed 10 to 1,000-fold increase in sensitivity compared to standard DNA extraction and modified PK digestion. No amplification was observed in samples prepared by boiling. The sample preparation of BHV-1 LA strain by GeneReleaserTM showed sensitivity equivalent to virus isolation. The BHV-1 TK PCR using GeneReleaserTM has a detection limit of 1 picogram and 10 fentograms of purified BHV-1 DNA using ethidium bromide stained gel and Southern blot hybridization, respectively. It could detect viral DNA in 1,000 infected cells in a total suspension of 10,000 cells using either ethidium bromide stained gel or Southern blot hybridization.

Amplification of strains of bovine herpesvirus 1 by use of polymerase chain reaction with primers in the thymidine kinase region.

A primer pair was designed from the published nucleotide sequence of the coding region of the bovine herpesvirus 1 (BHV-1) thymidine kinase (tk) gene for use in detection of the virus by use of polymerase chain reaction (PCR) amplification of 12 BHV-1 strains (3 ATCC and 9 local isolates). A tk deletion mutant BHV-1, and 2 BHV-4 strains from ATCC were used as negative controls. One strain each of feline herpes-virus, equine herpesvirus, and bovine adenovirus, and 2 noninoculated bovine cultured cells--bovine fetal testis and Madin-Darby bovine kidney--also were examined to verify specificity of the primers. A PCR product, 183 bp long, was detected by ethidium bromide staining after agarose gel electrophoresis, when purified DNA from cell cultures infected with BHV-1 strain LA was used as template. Specificity of the PCR product was confirmed by restriction digestion with Sac II enzyme and Southern blot hybridization. Amplification was detected by ethidium bromide staining of agarose gels and/or Southern blot hybridization with the radiolabeled PCR product of the LA strain in similarly prepared DNA templates of 5 other BHV-1 strains, 2 obtained from ATCC and 3 of the 9 local isolates. In a modified PCR protocol, using virus suspensions treated with a nucleic acid-releasing cocktail, substantial amplification was obtained for the 3 BHV-1 strains from ATCC and for all 9 local bovine herpesvirus field isolates.(ABSTRACT TRUNCATED AT 250 WORDS)

Proventricular impaction associated with nonsuppurative encephalomyelitis and ganglioneuritis in two Canada geese.

Two wild Canada geese (Branta canadensis) in an extremely emaciated state and with severe proventricular food impaction also had a nonsuppurative encephalomyelitis and ganglioneuritis. The condition in these two birds was morphologically similar to psittacine proventricular dilatation, a recently identified disease of psittacine birds.

The use of ELISA for detection of the antibody-induced conformational change in a viral protein and its intermolecular spread.

Antibody-induced conformational changes of proteins have been recently frequently suggested to explain a variety of observations. In spite of the fundamental importance of this phenomenon for both in vivo and in vitro antigen-antibody interactions, it is not generally accepted because of the lack of conclusive evidence. This report utilizes a novel approach to the study of antibody-induced antigenic conformational changes. Pairs of monoclonal antibodies (mAb) were used to induce and to assess conformational changes in potato virus X (PVX) protein. Blocking ELISA with native and glutaraldehyde treated virus was used to detect conformational changes. Double antibody sandwich (DAS) ELISA was designed to investigate possible inter-molecular spread of conformational changes. Detection of one way blocking in a blocking ELISA, with a pair of mAbs reacting to non-overlapping epitopes, suggested conformational change as the mechanism of blocking. The putative conformational change was confirmed when the one way blocking was prevented using conformationally restrained virus. Inter-molecular spread of the conformational change among the molecules of PVX protein was demonstrated in DAS-ELISA, when capture mAb inhibited binding of detecting mAb in the absence of steric hindrance. Unlike X-ray crystallography, the methodology utilized in this study indicates directly the significance of a changed conformation to antibody binding.

Pathogenesis of rotavirus infection in various age groups of chickens and turkeys: pathology.

Various age groups of turkeys, White Leghorn chickens, and broiler chickens were inoculated with turkey rotavirus strain Tu-2 or with chicken rotavirus Ch-2, and the development of rotavirus-induced lesions were evaluated macroscopically and microscopically (light microscopy and scanning electron microscopy). Morphometric evaluations were conducted to determine morphologic changes in the villi of infected turkeys. Macroscopic lesions that were found in turkeys, but not in chickens, consisted of pallor of the intestinal tract and distension of the cecum with frothy or nonfrothy fluid contents. Histologic lesions in turkeys consisted of basal vacuolation of enterocytes, separation of enterocytes from the lamina propria (with subsequent desquamation), villus atrophy accompanied by widening of the lamina propria, scalloping of the villus surface, fusion of the villi, and leukocytic infiltration of the lamina propria. Scanning electron microscopy indicated roughened villus surfaces, distortion of the normal morphologic features of the villi, and loss of microvilli in cells located on the tips of the villi. Most of the lesions disappeared by 8 days after inoculation. Results of the morphometric evaluations indicated that the crypt length had increased and the villus-to-crypt ratio had significantly decreased, compared with that of noninoculated control turkeys. Broilers greater than or equal to 21 days old and White Leghorn chickens greater than or equal to 35 days old had minimal leukocytic infiltration of the lamina propria and minimal loss of microvilli in cells located on the tips of the villi. The loss of microvilli was more extensive in chickens greater than or equal to 119 days old than in younger birds. Generally, turkeys 1 to 112 days old developed more severe lesions than did chickens, and lesions were more pronounced in turkeys at 112 days of age.

Pathogenesis of rotavirus infection in various age groups of chickens and turkeys: clinical signs and virology.

Age-related susceptibility patterns of turkeys, broilers, and specific pathogen-free (SPF) White Leghorn chickens to experimentally induced infection with turkey or chicken rotavirus isolates were compared. The following determinants were evaluated: clinical signs, onset and duration of virus production, viral titers, involvement of intestinal villi in the replication of the virus, and the development of antibodies against the virus. Older turkeys and chickens were more susceptible than were their younger counterparts, turkeys were more susceptible than were broiler and White Leghorn chickens (regardless of age), and broiler chickens were slightly more susceptible than were age-matched White Leghorn chickens. Turkeys developed diarrhea, accompanied by high viral titers within 1 day after inoculation with virus. Viral antigen was found in the epithelial cells of the intestinal villi throughout the intestinal tract and some cells of the cecal tonsils. Antibodies could be detected as early as 4 to 5 days after inoculation. These findings were more pronounced in turkeys inoculated at 112 days of age than in birds inoculated at a younger age. Age-related susceptibility patterns were similar in White Leghorn and broiler chickens. Infection was subclinical in birds less than 56 days old, whereas older birds developed soft feces. Egg production in the White Leghorn chickens decreased after being inoculated with virus at 350 days of age.

Experimental infection of specific-pathogen-free chickens with avian rotaviruses.

One-to-70-day-old specific-pathogen-free chickens were infected with rotaviruses isolated from chickens, turkeys, and pheasants. Chicks infected at 1 or 7 days of age remained largely free of infection: virus could be isolated from a few chicks of these ages, but virus antigen could not be detected in the epithelial cells of the intestinal villi. Treating the virus with trypsin before inoculation did not markedly enhance virus infectivity. The same batches of virus successfully infected chickens at 56 and 70 days of age: virus could be isolated between 1 and 5 days postinfection, and virus antigen was detected in the epithelial cells of the intestinal villi. Rotaviruses isolated from pheasants were infectious for 49-day-old chickens. All infections in the older chickens remained subclinical.

Pathogenesis of rotavirus infection in turkey poults.

The pathogenesis of rotavirus infection was examined after experimental infection of conventional and specific-pathogen-free (SPF) turkey poults. In six experiments birds were exposed to turkey rotavirus isolates Tu-1 or TU-2 or the chicken isolate Ch-1 at 7, 10 or 42 days of age. Poults were examined between 1 and 24 days post-infection (dpi) for diarrhoea, gross and histopathologic lesions, virus excretion in the intestinal tract, viral antigen in intestinal epithelial cells, and the development of serum antibodies. Between 2 and 5 dpi watery droppings were observed in conjunction with remarkable paleness of the intestinal tract which was grossly observable. Maximum viral replication occurred between 2 and 5 dpi, during which period viral antigen could be demonstrated in the epithelial cells of the duodenum, jejunum, ileum and colon. Sporadically, virus antigen-positive cells were seen in the cecum. As early as 4 to 5 dpi rotavirus antibodies could be detected by indirect immunofluorescence assays. Remarkable leukocyte infiltration of the lamina propria, vacuolation of the epithelial cells and scalloping of the villous surface at the tips were observed in the intestine of infected birds. Infection with rotavirus caused a significant impairment at 2 and 4 dpi of absorption of D-xylose from the intestinal tract.