Detection and molecular characterization of astro and bocaviruses in dogs in Minnesota.

Canine astrovirus (CAstV) and canine bocavirus (CBoV) are involved in cases of mild, and sometimes severe, gastroenteritis in dogs. Fecal samples from two dead dogs with gastroenteritis were received at the University of Minnesota Veterinary Diagnostic Laboratory to determine the cause of death. Small round viruses of 20-35 nm diameter were observed by negative contrast electron microscopy. The samples were subjected to Illumina MiSeq sequencing. Both samples were strongly positive for CAstV; all viral reads were related to CAstV. In addition, sample number 1 had a few reads of CBoV. Two complete sequences of CAstV were identified (6625 and 6627 nt in length) with 95% nt identity. RT-PCR and PCR were used to confirm CAstV and CBoV infections in successive samples of canine gastroenteritis. Sanger sequencing was done on nucleic acids from positive samples. Of a total of ten samples, CAstV and CBoV infections were confirmed in six and eight animals, respectively. Four animals had mixed infection with both viruses. All sequences of ORF1b gene of CAstVs showed closest clusters in phylogenetic tree with 96-100% nucleotide and amino acids identity. On the other hand, identity between VP2 gene of different CBoV strains in this study ranged from 93%- 100%. All strains were located close to each other except the divergent MT078234 strain, which was arranged in a separate branch and was closer to reference strain JN648103/USA/2010. This study highlights the importance of electron microscopy and next generation sequencing for early detection and characterization of viruses associated with dog gastroenteritis.

Comparative growth kinetic study of Newcastle disease virus, infectious bursal disease virus and avian influenza virus in chicken embryo fibroblast and DF-1 cell lines.

Viral diseases have caused devastating effect on poultry industry leading to significant losses in economy of world. In the presented study, the ability of Newcastle disease virus (NDV), infectious bursal disease virus (IBDV) and avian influenza virus (AIV) to grow in two cell lines was evaluated. Both chicken embryo fibroblast (CEF) and DF-1 cells were used and cytopathic effects (CPE) produced by these viruses were observed. The titer of virus in terms of TCID was determined after 24h up to four days for each virus. The same type of CPE was observed for all virus- es used in the study in both DF-1 and CEF cells. IBDV showed CPE causing rounding of cells while NDV caused formation of multicellular large nuclei, cell fusion and rounding of cells. Giant cells with inclusions and aggregation of cells with intact monolayer was observed for AIV. In growth kinetic study, higher titer of IBDV and NDV was observed in CEF cells than DF-1 cells while for AIV, DF-1 cells showed higher titer than CEF cells. These results would be useful for furthers comparative studies on growth of different cell lines of various viruses to find a suitability for vaccine production.

Prevalence of parvovirus in Minnesota turkeys.

Poult enteritis syndrome (PES) is characterized by enteritis and decreased body weight gain in growing turkey poults between one d and 7 wk of age. Another syndrome called light turkey syndrome (LTS) causes a decrease in body weight of adult tom turkeys in Minnesota leading to huge economic losses. Reovirus, rotavirus, and astrovirus have been found in LTS and PES flocks in Minnesota. We tested 80 fecal pools collected from four LTS flocks and 35 fecal pools from non-LTS flocks for the presence of parvovirus. In addition, 116 fecal and meconium samples from turkeys submitted to the Minnesota Veterinary Diagnostic Laboratory (MVDL) also were tested. The samples were tested by PCR using primers for the non-structural 1 (NS1) gene of parvovirus. Of the 80 samples from LTS flocks, 41 were positive for parvovirus while 20 of 35 samples from non-LTS flocks were positive. The prevalence of parvovirus in fecal samples submitted to MVDL was relatively low; only five of the 116 pools were positive. The partial NS1 gene sequences from LTS and non-LTS samples showed 98 to 100% nt identity except for one divergent turkey parvovirus (TuPV) strain that revealed 90% identity and clustered with chicken-like parvoviruses. The presence of this divergent strain suggests circulation of a recombinant strain of TuPV in Minnesota turkeys. Our results indicate that TuPVs are circulating in both LTS and non-LTS flocks of turkeys in Minnesota, and further experimental studies are indicated to study the role of TuPV in LTS.

Isolation and molecular characterization of bovine enteroviruses in Egypt.

Enteroviruses belong to the Picornaviridae family and infect a wide range of mammals including cattle. Bovine enterovirus (BEV) has recently been reclassified into E and F serotypes. BEV was first isolated in Egypt in 1966 although it has been known in other countries since the 1950s. In this study, BEV-F2 was isolated from calves with severe diarrhea and the isolated viruses were subjected to molecular characterization. Illumina sequencing of one of the isolates revealed the presence of a complete BEV-F genome sequence. The phylogenetic analysis revealed nucleotide substitutions along the genome in comparison with other known strains of BEV-F (HQ663846, AY508697 and DQ092795). Two primer sets were designed from the 3D and 5'NTR regions and used for the examination of the remaining isolates, which were confirmed to be of the BEV-F2 serotype. The availability of the complete genome sequence of this virus adds to the sequence database of the members of Picornaviridae and should be useful in future molecular studies of BEV.

Evaluating the virucidal efficacy of hydrogen peroxide vapour.

BACKGROUND: Surface contamination has been implicated in the transmission of certain viruses, and surface disinfection can be an effective measure to interrupt the spread of these agents. AIM: To evaluate the in-vitro efficacy of hydrogen peroxide vapour (HPV), a vapour-phase disinfection method, for the inactivation of a number of structurally distinct viruses of importance in the healthcare, veterinary and public sectors. The viruses studied were: feline calicivirus (FCV, a norovirus surrogate); human adenovirus type 1; transmissible gastroenteritis coronavirus of pigs (TGEV, a severe acute respiratory syndrome coronavirus [SARS-CoV] surrogate); avian influenza virus (AIV); and swine influenza virus (SwIV). METHODS: The viruses were dried on stainless steel discs in 20- or 40-µL aliquots and exposed to HPV produced by a Clarus L generator (Bioquell, Horsham, PA, USA) in a 0.2-m(3) environmental chamber. Three vaporized volumes of hydrogen peroxide were tested in triplicate for each virus: 25, 27 and 33 mL. FINDINGS: No viable viruses were identified after HPV exposure at any of the vaporized volumes tested. HPV was virucidal (>4-log reduction) against FCV, adenovirus, TGEV and AIV at the lowest vaporized volume tested (25 mL). For SwIV, due to low virus titre on the control discs, >3.8-log reduction was shown for the 25-mL vaporized volume and >4-log reduction was shown for the 27-mL and 33-mL vaporized volumes. CONCLUSION: HPV was virucidal for structurally distinct viruses dried on surfaces, suggesting that HPV can be considered for the disinfection of virus-contaminated surfaces.

In vitro characterization of influenza A virus attachment in the upper and lower respiratory tracts of pigs.

The binding of influenza A viruses to epithelial cells in the respiratory tract of mammals is a key step in the infection process. Therefore, direct assessment of virus-host cell interaction using virus histochemistry (VH) will enhance our understanding of the pathogenesis of these new viruses. For this study, the authors selected viruses that represented the 4 main genetic clusters of North American swine H1 (SwH1) viruses, along with A/California/04/2009 H1N1 and a vaccine strain for the positive controls, and the virus label, fluorescein isothiocyanate (FITC), for the negative control. A group of 5 viruses containing a 2-amino acid insertion adjacent to the binding site of the hemagglutinin protein and their presumed ancestral viruses were also examined for changes in binding patterns. Viruses were bound to formalin-fixed paraffin-embedded, 6-week-old (6w) and adult pig tissues. Qualitative VH scores per respiratory zone ranged from + to +++, with bronchioles having the highest and most consistent scores, regardless of animal age. For the 6w bronchioles, a quantitative VH score was calculated using digital images of 5 bronchioles per tissue section using image analysis software. Significant differences in attachment were found among the SwH1 viruses (P < .0001) and among the ancestral and insertion viruses (P < .0001). These results provide new insights on virus binding to porcine respiratory epithelial cells and the usefulness of morphometric scores. The results also highlight limitations of in vitro techniques, including VH for predicting virulence and host range.

The role of type-2 turkey astrovirus in poult enteritis syndrome.

An experimental study was conducted to determine the comparative pathogenicity of type-2 turkey astrovirus (TAstV-2) obtained from turkey flocks afflicted with poult enteritis syndrome (PES) and from turkey flocks displaying no apparent signs of infection. In total, ninety 7-d-old poults, which tested negative for the presence of astrovirus, rotavirus, coronavirus, and reovirus by reverse transcriptase (RT) PCR , were divided evenly into 3 groups: A, B, and C. Birds in group A were inoculated orally with turkey astrovirus-positive intestinal contents from birds affected with PES. Group B received turkey astrovirus-containing intestinal contents from apparently healthy flocks. Group C served as a negative control and was given PBS. Clinical signs of diarrhea, depression, and dullness were observed in group A. Birds in group B also showed clinical signs similar to those in group A, although the signs were milder in nature. Birds in group C did not show any clinical signs. At 16 d postinoculation, the BW of birds in group A was significantly lower than that of birds in groups B or C. In addition, the bursa size was reduced in group A, but not in groups B or C. Birds in groups A and B, but not in group C, were found to shed turkey astrovirus in their feces, as detected by RT-PCR. These results provide a preliminary indication that TAstV-2 from PES birds may be more pathogenic than TAstV-2 from apparently healthy poults. Further studies are needed to determine if pathogenic and nonpathogenic strains of TAstV-2 exist in the environment. These results also reinforce our previous observations that astrovirus is involved in PES, causing significant retardation in growth and weight gain.

Comparison of capsid gene sequences of turkey astrovirus-2 from poult-enteritis-syndrome-affected and apparently healthy turkeys.

This study was conducted to determine genetic variations in the capsid gene of turkey astrovirus-2 (TAstV-2) detected in apparently healthy and poult enteritis syndrome (PES)-affected turkeys. Capsid genes of astroviruses obtained from 30 PES-affected and 45 apparently healthy turkey flocks had sequence homologies of 73.4-100% and 72.4-100% at the nucleotide levels, respectively. The analysis of deduced amino acid sequences revealed one amino acid deletion at position 552 in 28 (93.3%) of 30 PES-affected cases. However, there were two deletions (at positions 551 and 552) in 31 (68.9%) of 45 TAstV-2 from apparently healthy flocks. The TAstV-2 (6.7%) from two PES-affected cases had two amino acid insertions each between positions 552 and 553, while TAstV-2 from 14 (31.1%) of 45 healthy flocks had two insertions at the same position. Phylogenetic analysis based on nucleotide sequences revealed that the astroviruses in this study were closely related to most of the previously published TAstV-2 isolates. The sequence homology of TAstV-2 in this study ranged from 70.4% to 99.4% at the nucleotide level with those of previously published TAstV-2 isolates. The variations at the amino acid level in the capsid gene suggest the possibility of the existence of different serotypes of turkey astrovirus. The close relationship of turkey astroviruses from apparently healthy flocks to those from PES-affected cases in capsid gene phylogeny necessitates further studies to compare complete capsid gene sequences from both types of flocks from different geographic areas for better understanding of TAstV circulating in turkeys.

Detection and molecular characterization of enteric viruses from poult enteritis syndrome in turkeys.

This study was conducted to detect and characterize enteric viruses [rotavirus, turkey astrovirus-2 (TAstV-2), reovirus, and turkey coronavirus] from cases of poult enteritis syndrome (PES) in Minnesota turkeys. Of the intestinal contents collected from 43 PES cases, 25 were positive for rotavirus and 13 for small round viruses by electron microscopy (EM). Of the enteric virus-positive cases by EM (n=27), 16 cases had rotavirus or small round viruses alone and the remaining 11 cases had both viruses. None of the cases were positive for reovirus or coronavirus by EM. However, with reverse transcription-PCR (RT-PCR), 40 cases (93%) were positive for rotavirus, 36 (84%) for TAstV-2, and 17 (40%) for reovirus. None of the cases were positive for turkey coronavirus by RT-PCR. The viruses from all cases were detected either alone or in combination of 2 or 3 by RT-PCR. Thus, 8 (19%) cases were positive for a single virus, whereas a combination of viruses was detected in the remaining 35 (81%) cases. The rota-TAstV-2 combination was the most predominant (n=18 cases). Fifteen cases were positive for all 3 viruses. The rotaviruses had sequence homology of 89.8 to 100% with previously published sequences of turkey rotaviruses at the nucleotide level. The TAstV-2 had sequence homology of 84.6 to 98.7% with previously published TAstV-2, whereas reoviruses had sequence homology of 91.6 to 99.3% with previously published sequences of turkey reoviruses. Phylogenetic analysis revealed that rota- and reoviruses clustered in a single group, whereas TAstV-2 clustered in 2 different groups. In conclusion, a larger number of PES cases was positive for rotavirus, TAstV-2, and reovirus by RT-PCR than with EM. The presence of more than one virus and changes at the genetic level in a virus may affect the severity of PES in turkey flocks.

Experimental reproduction of poult enteritis syndrome: clinical findings, growth response, and microbiology.

Poult enteritis syndrome (PES) is an infectious disease of turkey poults characterized by diarrhea, dullness, and depression. Five experiments were conducted to reproduce the disease in turkey poults using intestinal contents of PES-affected birds. In all experiments, poults at 14 d of age were divided into 4 groups and were orally given 2 mL of unfiltered supernatant, filtered supernatant, sediment dissolved in PBS, or PBS alone. Inocula in experiments 1, 3, and 5 consisted of intestinal contents from PES-affected birds of less than 2 wk of age, whereas those in experiments 2 and 4 consisted of intestinal contents from PES-affected birds of 4 to 6 wk of age. Poults in all groups were observed daily for clinical signs. The BW and microbiological criteria in experiments 1, 3, and 5 were evaluated at 5, 10, and 15 d postinoculation, whereas in experiments 2 and 4, these observations were made at 10 and 20 d postinoculation. Rotavirus, astrovirus, and Salmonella were present in all 5 inocula. Diarrhea and depression were the major signs in poults given PES material. Significant retardation of growth was observed in poults given any of the 3 PES materials, but this effect was more pronounced in poults given the sediment inoculum. Rotavirus, astrovirus, and Salmonella were detected in poults given PES material. In some cases, enterovirus was also detected. No major difference was noticed in experimental reproduction of PES when intestinal contents from different age birds were used as the inoculum.

In-vivo efficacy of hand sanitisers against feline calicivirus: a surrogate for norovirus.

Hand disinfection is considered important in preventing the transmission of viruses, including norovirus. We investigated the virucidal efficacy of nine hand sanitisers (four alcohol-based sanitisers, three non-alcoholic sanitisers and two triclosan-containing antimicrobial liquid soaps) against feline calicivirus, a surrogate for norovirus, on artificially contaminated fingertips for 30 s and 2 min contact periods. Among alcohol-based sanitisers, a product containing 99.5% ethanol was more effective than those containing 62% ethanol, 70% isopropanol or 91% isopropanol. A log(10) virus reduction factor of 1.00-1.30 was achieved with 99.5% ethanol but those containing a lower alcohol concentration only achieved a log(10) reduction factor of

Disinfection of fabrics and carpets artificially contaminated with calicivirus: relevance in institutional and healthcare centres.

Fabrics and carpets are used widely as surface coverings or linens in healthcare settings and are prone to contamination with infectious agents such as noroviruses (NoVs). Laundering, water cleaning and vacuuming are considered to be adequate for routine cleaning of these materials, but no standard procedure for their disinfection is available in case of contamination. Testing disinfectants for their efficacy against NoVs is difficult because these viruses cannot be cultivated in vitro. Therefore, feline calicivirus (FCV) has gained acceptance as a surrogate model for NoVs in disinfectant efficacy testing. The present study evaluated five disinfectants against FCV on various fabrics or carpets. FCV was dried on fabrics and carpets, followed by treatment with a given disinfectant for a defined contact time of 1, 5 or 10 min. The surviving virus was then eluted and titrated in Crandell-Reese feline kidney cells to determine virus inactivation. A disinfectant was considered to be effective if it inactivated at least 99% of the applied virus. Metricide, an activated dialdehyde-based product, was found to be the most effective disinfectant on all types of fabric and carpet, inactivating more than 99.99% of the virus in 1-10 min. In general, effectiveness of disinfectants increased with an increase in exposure time from 1 to 10 min. The disinfection of carpets was more difficult than the disinfection of fabrics; 100% polyester was the least amenable to disinfection. Only Metricide and Microbac-II (a phenolic compound) were able to inactivate 99% of FCV on 100% polyester. In summary, activated dialdehyde was found to be uniformly active against FCV on all types of material tested.

Pathogens and manure management systems: a review.

There has been an increasing concern about the effects of pathogens that are present in animal manure on humanand animal health. In recent years, outbreaks of food-borne diseases associated with the consumption of animal products havereceived much attention from the media in North America and Europe, leading to increased consumer concerns about the safety of their food supply. The health risks associated with animal operations depend on various factors. The most important ones appear to be related to the animal species being reared and the concentration of pathogenic microorganisms in animal manure. The ability of the pathogens to survive for long periods and through treatment to remain infective in the environment until ingested by human or animal host is an added concern. On the other hand, the role of livestock in most waterborne bacterial outbreaks has often been difficult to clarify since both humans and various wildlife species can shed the same microorganisms and thereby serve as sources of infection. This paper summarizes existing information on the main microbial pathogens present in livestock wastes, and discusses the impact of livestock wastes and agricultural drainage on microbiological quality of water, as well as available management and treatment technologies to minimize the prevalence of pathogens in animal wastes. Despite the fact that most disease outbreaks have been associated with food poisoning by cross-contamination during meat or milk processing and during finished product storage this review shows that a number of best management practices and technical solutions have been developed in the last few years that can be effective tools in minimizing the spread of pathogens from livestock operations in the environment.

Prevalence of swine influenza virus subtypes on swine farms in the United States.

Serologic and virologic prevalence of infection with different swine influenza virus (SIV) subtypes was investigated using swine sera, nasal swabs and lung samples that had been submitted for a diagnosis to the Minnesota Veterinary Diagnostic Laboratory. A total of 111,418 pig sera were tested for SIV antibody between 1998 and 2000, and 25,348 sera (22.8%) were found to be positive by the hemagglutination inhibition (HI) test. Of the positive samples, 16,807 (66.7%) and 8,541 (33.7%) had antibody to H1 and H3 subtypes, respectively. Between January 1998 and May of 2001, a total of 3,561 nasal swabs or lung samples were examined for the presence of SIV, and SIV was isolated from 1,124 samples (31.7%). Of these isolates, 869 (77.3%) and 255 (22.7%) were subtyped as H1 and H3, respectively, by the HI method. For further characterization, 120 SIV isolates each from 1998 to 2001 were randomly selected from a culture collection and their hemagglutinin (HA) and neuraminidase genes examined by reverse transcription-PCR and sequencing. Of the 480 isolates, 322 (67.1%), 22 (4.6%) and 129 (26.9%) were subtyped as H1N1, H1N2 and H3N2, respectively. The remaining 7 samples (1.5%) were found to contain both H1N1 and H3N2 viruses. The SIV H1N2 subtype was isolated from 1, 8, and 13 samples in 1999, 2000, and 2001, respectively. The 22 H1N2 isolates originated from 9 different states of the United States. Genetic screening of the HA genes of 12 selected H1N2 isolates showed that 8 of them had a close phylogenetic relationship with the Indiana isolate of H1N2 (A/Swine/Indiana/9K035/99), while 4 isolates were closely related to classical SIV H1N1.

Detection and subtyping of swine influenza H1N1, H1N2 and H3N2 viruses in clinical samples using two multiplex RT-PCR assays.

A total of 360 type A swine influenza virus-positive samples including cell culture isolates, nasal swabs or lung tissues along with 30 virus-negative samples were tested for the detection and subtyping of H1N1, H1N2 or H3N2 by two multiplex reverse transcription (RT)-PCR assays. The positive samples had been collected between 1999 and 2001 from pigs with respiratory diseases, and type A influenza virus was isolated and subtyped by hemagglutination inhibition (HI) test at the Minnesota Veterinary Diagnostic Laboratory (MVDL). Two multiplex RT-PCR assays specific for H1 and H3, and N1 and N2 were developed. RT-PCR products with unique sizes characteristic of each subtype of influenza A virus were sequenced, and the sequences were demonstrated to be specific for H1N1, H1N2 or H3N2. Genomic RNAs or DNAs from 12 common swine pathogens other than type A influenza viruses were not amplified when the PCR assays were performed with these primer sets. Positive amplification reaction could be visualized with RNA extracted from up to 10(-5) dilution of each reference virus with original infectivity titer of 10(5) TCID(50)/ml. Of the 360 samples tested, swine influenza virus H1N1, H1N2 and H3N2 were identified in 200, 13 and 139 samples, respectively. The remaining eight samples were positive for both H1N1 and H3N2 viruses. The results of multiplex RT-PCR were 100% in agreement with those of virus isolation. These results demonstrate the usefulness of multiplex RT-PCR for detection and identification of influenza A virus subtypes. The results also indicate an increased occurrence of H1N2 in US swine population.

Sequence analysis of the nucleocapsid and phosphoprotein genes of avian pneumoviruses circulating in the US.

Avian pneumovirus (APV) has recently been described as the cause of a new respiratory syndrome in turkey flocks in the United States. We here describe the complete sequence of the nucleocapsid (N) and phosphoprotein (P) genes of this emerging APV (APV/US). Our results show 59 and 61% nucleotide sequence identity of the APV/US N gene with N genes of previously described European APV subgroups A and B, respectively. The P gene of APV/US showed only 53% nucleotide sequence identity with the ortholog from APV subgroup A. Phylogenetic analyses of both N and P genes clearly demonstrate that the APV/US lineage is evolutionarily related but distinct from European APVs. Moreover, sequence analysis of the N and P genes from two laboratory adapted isolates of APV/US (APV/MN-1a and APV/MN-1b) and from ten clinical samples from APV-infected turkeys suggests only modest level of amino acid divergence in the N (0-0.3%) and P (0-1.4%) proteins. Taken together, the results of this study indicate support that APV/US represents a new subgroup (subgroup C) of APV and show that there is limited heterogeneity in the N and P genes of APV/US isolates.

Protective efficacy of high-passage avian pneumovirus (APV/MN/turkey/1-a/97) in turkeys.

A U.S. isolate of avian pneumovirus (APV), APV/MN/turkey/1-a/97, was attenuated by serial cell culture passages in chicken embryo fibroblasts (seven passages) and Vero cells (34 passages). This virus was designated as APV passage 41 (P41) and was evaluated for use as a live vaccine in commercial turkey flocks. The vaccine was inoculated by nasal and ocular routes in 2-to-4-wk-old turkeys in 10 turkey flocks, each with 20,000-50,000 birds. Only 2 birds per 1000 birds were inoculated in each flock with the expectation that bird-to-bird passage would help spread the infection from P41-exposed birds to their respective flock mates. The virus did spread from vaccinated birds to the entire flock within 10 days as detected by reverse transcription-polymerase chain reaction. Mild respiratory illness was observed in a few birds 12 days postvaccination in 2 of 10 flocks. Within 3 wk postvaccination, all flocks became seropositive for APV antibodies as measured by enzyme-linked immunosorbent assay. In an additional flock, the virus was administered to all turkeys simultaneously in drinking water and seroconversion occurred within 2 wk. All 11 flocks remained seropositive until 10 wk postvaccination. When compared with unvaccinated flocks on the same farm from the previous year, the medication cost, total condemnation, and mortality rates attributed to APV were lower in P41-vaccinated flocks. When birds from vaccinated flocks were challenged with virulent APV under experimental conditions, no clinical signs were observed at 2, 6, and 10 wk postvaccination, whereas in the control unvaccinated birds, respiratory illness and virus shedding occurred after challenge. These results indicate that P41 administered by the nasal and ocular routes, and by drinking water, causes seroconversion and induces protection from virulent APV challenge for at least 10 wk.

Efficacy of commonly used disinfectants for the inactivation of calicivirus on strawberry, lettuce, and a food-contact surface.

Norwalk and Norwalk-like viruses (NLVs) are important causes of foodborne gastroenteritis in restaurant-related outbreaks. Efficacy of common disinfection methods against these viruses on food-contact surfaces and fresh produce is not known partially because of their nonculturability. Seven commercial disinfectants for food-contact surfaces and three sanitizers for fruits and vegetables were tested against cultivable feline calicivirus (FCV). Disks of stainless steel, strawberry, and lettuce were contaminated with known amounts of FCV. The disinfectants were applied at one, two, and four times the manufacturer's recommended concentrations for contact times of 1 and 10 min. The action of disinfectant was stopped by dilution, and the number of surviving FCVs was determined by titration in cell cultures. An agent was considered effective if it reduced the virus titer by at least 3 log10 from an initial level of 10(7) 50% tissue culture infective dose. None of the disinfectants was effective when used at the manufacturer's recommended concentration for 10 min. Phenolic compounds, when used at two to four times the recommended concentration, completely inactivated FCV on contact surfaces. A combination of quaternary ammonium compound and sodium carbonate was effective on contact surfaces at twice the recommended concentration. Rinsing of produce with water alone reduced virus titer by 2 log10. On artificially contaminated strawberry and lettuce, peroxyacetic acid and hydrogen peroxide was the only effective formulation when used at four times the manufacturers' recommended concentration for 10 min. These findings suggest that FCV and perhaps NLVs are very resistant to commercial disinfectants. However, phenolic compounds at two to four times their recommended concentrations appear to be effective at decontaminating environmental surfaces and may help control foodborne outbreaks of calicivirus in restaurants.

PCR-based detection of an emerging avian pneumovirus in US turkey flocks.

Avian pneumovirus (APV) or turkey rhinotracheitis virus (TRTV) is an important respiratory pathogen of domesticated poultry in many countries in Europe, Africa, and Asia. Until recently, the United States was considered free of APV. In late 1996, an atypical upper respiratory tract infection appeared in turkey flocks in Colorado and shortly thereafter in turkey flocks in Minnesota. An avian pneumovirus (APV-US) that was serologically distinct from the previously described TRTV was isolated as the primary cause of the new syndrome. The nucleotide sequence of a fragment of the APV-US fusion gene was determined and used to develop a polymerase chain reaction-based assay that specifically detects APV-US viral nucleic acid sequences in RNA extracts of tracheal swabs and turbinate homogenates. The assay is highly sensitive in that it can detect <0.01 TCID50 of APV. The availability of this assay enables the rapid and accurate determination of APV-US in infected poultry flocks.

Detection of antibodies to U.S. isolates of avian pneumovirus by a recombinant nucleocapsid protein-based sandwich enzyme-linked immunosorbent assay.

The nucleocapsid (N) protein of subgroup C (United States-specific) avian pneumovirus (APV/US) was expressed in Escherichia coli, and antibodies to the recombinant N protein were shown to specifically recognize the approximately 47-kDa N protein of APV/US by Western immunoblot analysis. The recombinant APV/US N protein was used in a sandwich-capture enzyme-linked immunosorbent assay (ELISA), and the resulting assay was found to be more sensitive and specific than the routine indirect ELISA for the detection of APV/US antibodies in turkey sera.