Complete Genome Sequencing Reveals Unusual Equine Rotavirus A of Bat Origin from India.

Rotaviruses are the most common viral agents associated with foal diarrhea. Between 2014 and 2017, the annual prevalence of rotavirus in diarrheic foals ranged between 18 and 28% in Haryana (India). Whole-genome sequencing of two equine rotavirus A (ERVA) isolates (RVA/Horse-wt/IND/ERV4/2017 and RVA/Horse-wt/IND/ERV6/2017) was carried out to determine the genotypic constellations (GCs) of ERVAs. The GCs of both the isolates were G3-P[3]-I8-R3-C3-M3-A9-N3-T3-E3-H6, a unique combination reported for ERVAs so far. Both the isolates carried VP6 of genotype I8, previously unreported from equines. Upon comparison with RVAs of other species, the GC of both isolates was identical to that of a bat rotavirus strain, MSLH14, isolated from China in 2012. The nucleotide sequences of the genes encoding VP3, NSP2, and NSP3 shared >95.81% identity with bat RVA strains isolated from Africa (Gabon). The genes encoding VP1, VP2, VP7, NSP1, and NSP4 shared 94.82% to 97.12% nucleotide identities with the human strains which have zoonotic links to bats (RCH272 and MS2015-1-0001). The VP6 genes of both strains were distinct and had the highest similarity of only 87.08% with that of CMH222, a human strain of bat origin. The phylogenetic analysis and lineage studies revealed that VP7 of both isolates clustered in a new lineage (lineage X) of the G3 genotype with bat, human, and alpaca strains. Similarly, VP4 clustered in a distinct P[3] lineage. These unusual findings highlight the terra incognita of the genomic diversity of equine rotaviruses and support the need for the surveillance of RVAs in animals and humans with a "one health" approach. IMPORTANCE Rotaviruses are globally prevalent diarrheal pathogens in young animals including foals, piglets, calves, goats, sheep, cats, and dogs along with humans. The genome of rotaviruses consists of 11 segments, which enables them to undergo reshuffling by reassortment of segments from multiple species during mixed infections. In this study, the prevalence of equine rotaviruses was 32.11% in organized equine farms of North India. The complete genome analysis of two ERVA isolates revealed an unusual genomic constellation, which was previously reported only in a bat RVA strain. A segment-wise phylogenetic analysis revealed that most segments of both isolates were highly similar either to bat or to bat-like human rotaviruses. The occurrence of unusual bat-like rotaviruses in equines emphasizes the need of extensive surveillance of complete genomes of both animal and human rotaviruses with a "one health" approach.

Development of IgM-ELISA for diagnosis of recent infection of Japanese encephalitis virus in equines.

Japanese encephalitis (JE) is a re-emerging mosquito borne disease, for which equines are most susceptible amongst all animals. Detection of specific immunoglobulin 'M' (IgM) is considered as an ideal way to diagnose recent JE virus infection in equines due to low virus load and short-term viremia. The present study was undertaken to develop a sensitive and specific recombinant NS1 protein based indirect IgM-ELISA and IgM capture (MAC) ELISA to diagnose recent infection of JEV in equines. Indirect IgM ELISA was standardized with relative diagnostic sensitivity and specificity of 100% and 88.5%, respectively. The validation of indirect IgM-ELISA in different laboratories revealed excellent reproducibility with Cohen's kappa value ranging between 0.84 and 1. The standardization of MAC ELISA was attempted using checker board titration method and non-specific binding of polyclonal anti-equine IgM capture antibody with anti-porcine IgG conjugate and with hyperimmune serum raised in swine against the antigen was observed. Hence, the MAC ELISA was standardized with monoclonal capture antibody; however, its diagnostic performance could not meet the satisfactory limit. Due to better sensitivity and less turnaround time, indirect IgM-ELISA was employed to screen 821 equine serum samples revealing 33.73% positivity of IgM antibodies against JEV in equine population of India. The high JEV sero-positivity warrants the need for vaccination in Indian equine population along with the demand for research focused towards anti-viral therapy. The indirect IgM-ELISA developed in the present study could be useful to diagnose acute or recent infection of JEV in equines as well as in sero-epidemiological studies.

Development of recombinant nonstructural 1 protein based indirect enzyme linked immunosorbent assay for sero-surveillance of Japanese encephalitis in swine.

Japanese encephalitis virus (JEV) causes severe neurological disease in humans, especially among children. The disease is endemic in several South Asian countries including India. Swine play a major role as amplifier host for JEV and act as a source of infection to humans through mosquito bite. Early detection of either virus or antibodies in swine will aid to undertake control measures to prevent virus spread to humans. Swine seldom show symptoms of JEV infection and the viraemic phase lasts for a short period of 3 to 4 days indicating the potential of detection of antibodies, which remain for relatively longer period, as a suitable alternative. Cost effective and sensitive assays for the detection of JEV antibodies in swine are not available indigenously. Hence, we have developed a recombinant nonstructural protein 1 (rNS1) based enzyme linked immunosorbent assay for the detection of IgG antibodies against JEV in swine. The test is robust, highly sensitive (91%), specific (97%), reproducible and affordable. Field validation of the assay was done by screening 3628 swine Serum samples collected from different parts of India. The overall sero-positivity was found to be 32.22%. The developed ELISA can be readily incorporated into surveillance programs for detection of Japanese encephalitis virus activity in swine population thereby aiding in prediction of outbreaks in humans.

Zoonotic Viral Diseases of Equines and Their Impact on Human and Animal Health.

INTRODUCTION: Zoonotic diseases are the infectious diseases that can be transmitted to human beings and vice versa from animals either directly or indirectly. These diseases can be caused by a range of organisms including bacteria, parasites, viruses and fungi. Viral diseases are highly infectious and capable of causing pandemics as evidenced by outbreaks of diseases like Ebola, Middle East Respiratory Syndrome, West Nile, SARS-Corona, Nipah, Hendra, Avian influenza and Swine influenza. EXPALANTION: Many viruses affecting equines are also important human pathogens. Diseases like Eastern equine encephalitis (EEE), Western equine encephalitis (WEE), and Venezuelan-equine encephalitis (VEE) are highly infectious and can be disseminated as aerosols. A large number of horses and human cases of VEE with fatal encephalitis have continuously occurred in Venezuela and Colombia. Vesicular stomatitis (VS) is prevalent in horses in North America and has zoonotic potential causing encephalitis in children. Hendra virus (HeV) causes respiratory and neurological disease and death in man and horses. Since its first outbreak in 1994, 53 disease incidents have been reported in Australia. West Nile fever has spread to many newer territories across continents during recent years.It has been described in Africa, Europe, South Asia, Oceania and North America. Japanese encephalitis has expanded horizons from Asia to western Pacific region including the eastern Indonesian archipelago, Papua New Guinea and Australia. Rabies is rare in horses but still a public health concern being a fatal disease. Equine influenza is historically not known to affect humans but many scientists have mixed opinions. Equine viral diseases of zoonotic importance and their impact on animal and human health have been elaborated in this article. CONCLUSION: Equine viral diseases though restricted to certain geographical areas have huge impact on equine and human health. Diseases like West Nile fever, Hendra, VS, VEE, EEE, JE, Rabies have the potential for spread and ability to cause disease in human. Equine influenza is historically not known to affect humans but some experimental and observational evidence show that H3N8 influenza virus has infected man. Despite our pursuit of understanding the complexity of the vector-host-pathogen mediating disease transmission, it is not possible to make generalized predictions concerning the degree of impact of disease emergence. A targeted, multidisciplinary effort is required to understand the risk factors for zoonosis and apply the interventions necessary to control it.

Phenotypical and functional characteristics of mesenchymal stem cells derived from equine umbilical cord blood.

Mesenchymal stem cells (MSCs) offer promise as therapeutic aid in the repair of tendon and ligament injuries in race horses. Fetal adnexa is considered as an ideal source of MSCs due to many advantages, including non-invasive nature of isolation procedures and availability of large tissue mass for harvesting the cells. However, MSCs isolated from equine fetal adnexa have not been fully characterized due to lack of species-specific markers. Therefore, this study was carried out to isolate MSCs from equine umbilical cord blood (UCB) and characterize them using cross-reactive markers. The plastic-adherent cells could be isolated from 13 out of 20 (65 %) UCB samples. The UCB derived cells proliferated till passage 20 with average cell doubling time of 46.40 ± 2.86 h. These cells expressed mesenchymal surface markers but did not express haematopoietic/leucocytic markers by RT-PCR and immunocytochemistry. The phenotypic expression of CD29, CD44, CD73 and CD90 was shown by 96.36 ± 1.28, 93.40 ± 0.70, 73.23 ± 1.29 and 46.75 ± 3.95 % cells, respectively in flow cytometry, whereas, reactivity against the haematopoietic antigens CD34 and CD45 was observed only in 2.4 ± 0.20 and 0.1 ± 0.0 % of cells, respectively. Osteogenic and chondrogenic differentiation could be achieved using established methods, whereas the optimum adipogenic differentiation was achieved after supplementing media with 15 % rabbit serum and 20 ng/ml of recombinant human insulin. In this study, we optimized methodology for isolation, cultural characterization, differentiation and immunophenotyping of MSCs from equine UCB. Protocols and markers used in this study can be employed for unequivocal characterization of equine MSCs.

Buffalo (Bubalus bubalis) term amniotic-membrane-derived cells exhibited mesenchymal stem cells characteristics in vitro.

Recent studies suggested that placentae amniotic membrane is a valuable source of stem cells in human as well as in livestock species. Advantages of amnion over other sources of stem cells included abundant availability, ethically non-objectionable and non-invasive source. The aim of the present study was the isolation, culture and characterization of amniotic-membrane-derived mesenchymal stem cells from term placentae collected postpartum in buffalo. We have observed that both presumptive epithelial-like and fibroblast-like cells were cultured and maintained from term amnion. These cells were shown the positive expression of pluripotency markers (OCT-4, SOX-2, NANOG, TERT), mesenchymal stem cell markers (CD29, CD44, CD105) and negative for haematopoietic marker (CD34) genes at different passages. In addition, these cells were also positive for alkaline phosphatase staining. Stem-ness potential of any stem cells is determined by their potential to differentiate into specific lineages of cell type. In the present study, we have successfully differentiated the amniotic-membrane-derived cells into adipogenic, chondrogenic and osteogenic lineages of cells in vitro. In conclusion, the results of this study demonstrate that amniotic-membrane-derived cells expressed pluripotent and mesenchymal stem cells markers and have propensity to differentiate into cells of mesenchymal lineage cell type upon directed differentiation in vitro.

Genetic Analysis of the Neuraminidase (NA) Gene of Equine Influenza Virus (H3N8) from Epizootic of 2008-2009 in India.

The neuraminidase (NA) gene sequences of four Indian equine influenza viruses (EIVs) isolated from epizootic in 2008 and 2009 were analyzed. The phylogenetic relationship and selection pressure of NA genes were established in comparison to other EIVs circulating worldwide along with the domains and motifs of the encoded protein to find out the significance of mutational changes. Among Indian isolates, two amino acid (aa) changes each in Mysore/12/08 (Asn67Tyr & Asp396Gly), Gopeshwar/1/09 (Ile49Val & Asp396Gly), and Uttarkashi/1/09 (Ile49Val & Asp396Gly) isolates were observed in respect to Jammu-Katra/06/08 isolate. Amino acid (aa) sequence analysis also revealed five consistent aa residue changes viz, Gly/Arg40Glu, Tyr66His, Val191Ile, Val209Ile and Asp235Asn in Asian including Indian isolates, Spain/07 and Spain/09 isolates in comparison to other EIVs circulating worldwide. The topology of the phylogenetic tree revealed that the Indian, Chinese, Mongolian and Kazakhstan isolates together formed a subgroup with Yokohama/10 isolate. Spain/07 & Spain/09 isolates showed closest clustering with Asian isolates. This indicates that non-synonymous mutations in Asian isolates with temporal pattern originating from Spain/07, led to the subgroup of the Asian isolates within Florida clade 2 sublineage. The analysis of the predicted secondary structure has not shown any significant difference in the NA proteins of all Indian isolates. Fixed-effects likelihood (FEL) analysis of the selection pressure revealed three codons (43, 355 & 434) under positive selection pressure. The overall evolutionary changes (ω value) of 3.4 indicates NA gene to be under strong selection pressure. Further, seven putative N-glycosylation sites were observed in the NA protein. The mapping of specific aa changes, their mutational and functional analysis need to be carried out to ascertain their role in pathogenecity of the virus.

Genetic analysis of the matrix and non-structural genes of equine influenza virus (H3N8) from epizootic of 2008-2009 in India.

India faced an epizootic of equine influenza in 2008-2009. The isolated viruses were typed as H3N8 and grouped with the clade 2 viruses of Florida sublineage on the basis of haemagglutinin (HA) gene sequence analysis. This report describes the genetic analysis and selection pressure of matrix (M) and non-structural 1 (NS1) genes of the Indian isolates. All isolates shared 98.41% and 99.54% homology with other clade 2 viruses of Asian origin for M1 and M2 amino acid (aa) sequences, respectively. There were 3 and 4 unique aa residue changes respectively in M1 and M2 proteins in all Asian isolates. Phylogenetic analysis revealed clustering of Indian and Chinese isolates in a separate group designated here as Asian clade for M gene. Indian and Chinese isolates shared homology ranging from 98.17% to 99.08% at aa level. The M and NS1 genes were under negative selection pressure with estimated magnitude of pressure (ω) 0.054, 0.581 and 0.30 for M1, M2 and NS1, respectively.

Zoonotic cases of camelpox infection in India.

This study reports the first conclusive evidence of zoonotic camelpox virus (CMLV) infection in humans associated with outbreaks in dromedarian camels (Camelus dromedaries) in northwest region of India during 2009. CMLV infection is usually restricted to camels and causes localised skin lesions but occasionally leads to generalised form of disease. However, the present outbreak involved camel handlers and attendants with clinical manifestations such as papules, vesicles, ulceration and finally scabs over fingers and hands. In camels, the pock-like lesions were distributed over the hairless parts of the body. On the basis of clinical and epidemiological features coupled with serological tests and molecular characterization of the causative agent, CMLV zoonosis was confirmed in three human cases. Clinical samples such as skin scabs/swabs and blood collected from affected animals and humans were analysed initially, for the presence of CMLV-specific antigen and antibodies by counter immunoelectrophoresis (CIE); serum neutralization test (SNT); plaque-reduction neutralization test (PRNT) and indirect immunoperoxidase test which was later confirmed by amplification of CMLV-specific ankyrin repeat protein (C18L) gene. Virus isolation was successful only from samples collected from camels. Further, sequence analyses based on three full-length envelope protein genes (A27L, H3L and D8L) revealed 95.2-99.8% and 93.1-99.3% homology with other Orthopoxviruses at nucleotide and amino acid levels, respectively. Phylogram of the three genes revealed a close relationship of CMLV with Variola virus (VARV). Considering the emerging and re-emerging nature of the virus, its genetic relatedness to VARV, zoonotic potential and productivity losses in camels; the control measures are imperative in curtailing economic and public health impact of the disease. This is the first instance of laboratory confirmed camelpox zoonosis in India.

Outbreak of Abortions and Infertility in Thoroughbred Mares Associated with Waterborne Aeromonas hydrophila.

At a thoroughbred equine breeding farm near Hissar (Haryana), three mares aborted in their seventh month of pregnancy. The vaginal swabs of all aborted mares, and stomach contents, heart blood, liver, spleen and placenta of aborted fetuses yielded pure culture of Aeromonas hydrophila. In addition, A. hydrophila was also isolated from the vaginal swabs of three repeat breeding mares and faecal sample of a diarrheic foal. The source of infection was possibly water supply as all the water samples collected from taps, mother tank and storage tank were found to be positive for A. hydrophila. The antibiogram of all the isolates was similar showing resistance to ampicillin, carbenicillin, gentamicin, kanamycin and amikacin but sensitive to chloramphenicol, ciprofloxacin, cefuroxime, ceftriaxone, cotrimoxazole, cotrimazine, nitrofurantoin, streptomycin and tetracycline. All the 20 sera samples collected from three aborted and three repeat breeding, and eight in-contact mares, a diarrheic foal, three cows and two male buffaloes maintained at the same farm contained antibodies against A. hydrophila with titres ranging from 80 to 640. The water supply was instantly chlorinated using 0.05% sodium hypochlorite for three consecutive days and all the culturally positive mares were treated with intravaginal administration of 1 g ciprofloxacin, while the foal was given nitrofurantoin for three days. After one month, A. hydrophila could not be isolated either from mares or from their environment and antibody titre in all the seropositive animals showed a declining trend. Later, all the aborted and repeat breeding mares were confirmed to be pregnant. Thus, the present study indicated that water-borne A. hydrophila might be associated with equine abortions and infertility, and diarrhea in newborn foals.

Monoclonal antibodies AC-43 and AC-29 disrupt Plasmodium vivax development in the Indian malaria vector Anopheles culicifacies (Diptera: Culicidae).

A repertoire of monoclonal antibodies (mAbs) was generated against the midgut proteins of Anopheles culicifacies mosquitoes. The mAbs AC-43 and AC-29 significantly inhibited Plasmodium vivax development inside the mosquito midgut. The number of oocysts that developed was reduced by 78.6% when mosquitoes ingested a combination of these two mAbs along with the blood meal. AC-43 mAb binds to the epitope common in 97, 80 and 43 kDa polypeptides from the midgut protein extract, as indicated by western blot analysis. Similarly, the mAb AC-29 recognized 52, 44, 40 and 29 kDa polypeptides. These female midgut-specific polypeptides are shared between An. culicifacies and An. stephensi, two major vectors of malaria in India. Deglycosylation assays revealed that O-linked carbohydrates are the major components in epitopes corresponding to AC-43 and AC-29. Gold particle labelling revealed that both these mAbs preferentially bind to glycoproteins at the apical microvilli and the microvillus-associated network present inside transverse sections of the gut epithelium. These regions are particularly known to have receptors for ookinetes, which enable them to cross this epithelial barrier and provide them with certain necessary chemicals or components for further development into oocysts. Therefore, these glycoproteins appear to be potential candidates for a vector-directed transmission-blocking vaccine (TBV).

Equine influenza outbreak in India (2008-09): virus isolation, sero-epidemiology and phylogenetic analysis of HA gene.

An outbreak of equine influenza (EI) was reported in India in June, 2008 after a gap of two decades. The outbreak started from Jammu and Kashmir (Katra), northern state of India and spread to the other parts of the country affecting equines in 11 states. The virus (H3N8) was isolated from nasal swabs obtained from clinical cases in various locations in the country including Katra (Jammu and Kashmir), Mysore (Karnataka) and Ahmedabad (Gujarat) using embryonated chicken eggs. The virus isolates were identified as H3N8 by haemagglutination inhibition (HI) test titration with standard serum and by sequencing of full-length haemagglutinin (HA) gene and partial sequence of neuraminidase (NA) gene. Paired serum samples (n=271) showing more than fourfold rise in antibody titres tested from 11 states confirmed equine influenza. Serum samples (n=2517) of equines from 13 states of the country screened by HI test revealed 687 (26.85%) samples positive for antibodies to EI (H3N8). Phylogenetic analysis of the haemagglutinin (HA) gene confirmed the virus to be closely related to Clade 2 of the Florida sublineage in American lineage. Comparison of deduced amino acid sequence of HA gene with EIV isolates from various lineages showed substitutions in the antigenic regions C and D. HA1 gene sequence had highest amino acid identity to A/eq/Gansu/7/08 and A/eq/Hubei/6/08 isolates from China and Inner-Mongolia isolate, while the complete HA gene sequence was closest to A/eq/A/eq/Newmarket/5/03, A/eq/Bari/05 and A/eq/Kentucky/05/02 isolates. Recent outbreaks of Mongolia, China and India by clade 2 EI viruses imply their predominance in Asia in addition to Europe.

Evaluation of immune response to bovine rotavirus following oral and intraperitoneal inoculation in mice.

With a view to use mice as an experimental model for studying immune response to bovine rotavirus (BRV), the kinetics of humoral and cellular immune responses to BRV in mice were evaluated by immunizing through intraperitoneal and oral route with UK strain of BRV. Following immunization with BRV, anti-rotavirus antibodies was developed in mice. The mean log antibody titres as measured by ELISA in mice immunized by intraperitoneal route were significantly higher than those immunized by oral route. Significant cellular immune response was observed in BRV-immunized mice on stimulation with BRV antigen, as measured by lymphocyte proliferation assay. The thymidine uptake by splenic and mesenteric lymph-node cells of intraperitoneally immunized mice on stimulation with BRV was 21328 +/- 1225 and 739 +/- 55 CPM, respectively. The splenic cells showed significantly higher stimulation (stimulation index 12.98) as compared to those of mesenteric cells (stimulation index 1.57). Foot pad inoculation test showed maximum virus-specific delayed type hypersensitivity reaction at 24 hr post-challenge following primary immunization and at 18 hr post-challenge following secondary immunization. The results indicate that BRV immunization by intraperitoneal route generates more efficient immune response in mice than by oral route and this route may be used for immune response studies involving BRV infection.

Diversity in Indian equine rotaviruses: identification of genotype G10,P6[1] and G1 strains and a new VP7 genotype (G16) strain in specimens from diarrheic foals in India.

Rotaviruses causing severe diarrhea in foals in two organized farms in northern India, during the period from 2003 to 2005, were characterized by electropherotyping, serotyping, and sequence analysis of the genes encoding the outer capsid proteins. Of 137 specimens, 47 (34.31%) were positive for rotavirus and exhibited at least five different electropherotypes (E), E1 to E5. Strains belonging to different electropherotypes exhibited either a different serotype/genotype specificity or a lack of reactivity to typing monoclonal antibodies (MAbs) used in this study. Strains belonging to E1, E2, and E5 exhibited genotype G10,P6[1], G3, and G1 specificities and accounted for 19.0, 42.9, and 9.5% of the isolates, respectively. Though they possessed G10-type VP7, the E1 strains exhibited high reactivity with the G6-specific MAb, suggesting that the uncommon combination of the outer capsid proteins altered the specificity of the conformation-dependent antigenic epitopes on VP7. E3 and E4 strains accounted for 28.6% of the isolates and were untypeable. Sequence analysis of VP7 from E4 strains (Erv92 and Erv99) revealed that they represent a new VP7 genotype, G16. The detection of unexpected bovine rotavirus-derived G10,P6[1] reassortants, G1 serotype strains, and a new genotype (G16) strain in two distant farms reveals an interesting epidemiological situation and diversity of equine rotaviruses in India.

Isolates of Theileria annulata collected from different parts of India show phenotypic and genetic diversity.

Phenotypic and genetic polymorphism was studied amongst four Theileria annulata isolates collected from three different parts of India. Amongst various markers studied for the comparison of growth characteristics of schizont cell lines established from these isolates, viability, non-viability counts and nitric oxide (NO) production showed significant variation. A negative correlation was observed between NO production and mRNA expression for TNF-alpha, a potent proinflammatory cytokine related to the pathogenesis of the disease. Phenotypic polymorphism was also revealed by T. annulata schizont-specific monoclonal antibodies (Mabs), viz. 1C7, 1E11, 2G2 and EU-106, which recognized variable number of cells in indirect fluorescent antibody and indirect immunoperoxidase tests, when tested against the four T. annulata isolates collected from India. Genetic polymorphism was recognized amongst the four isolates by restriction digestion analysis of Tams-1 gene PCR products. These observations revealed that the four isolates of T. annulata are different from each other and might be expressing different antigenic determinants on their cell surface.

Development of a neutralizing monoclonal antibody-based blocking ELISA for detection of equine herpesvirus 1 antibodies.

A single-dilution, sensitive and specific monoclonal antibody-based blocking enzyme-linked immunosorbent assay (B-ELISA) was developed as an alternative to the cumbersome virus neutralization test (VNT) for detection of equine herpesvirus-1 (EHV-1) antibodies. Neutralizing monoclonal antibodies (1H6 and 9C6) raised against EHV-1 (Hisar-90-7 strain) and sera from 70 horses (30 known negative and 40 known positive for EHV-1 antibodies by VNT) were used for standardization of the B-ELISA. Using a single serum dilution of 1:250 in B-ELISA, 100% specificity was obtained with both monoclonal antibodies (Mabs) in comparison to VNT. Similarly, the sensitivity of the B-ELISA was 92.5% and 100% with 1H6 and 9C6 Mabs, respectively. A very high correlation coefficient (r = 0.85) was observed between B-ELISA and VNT that was significant at the p < 0.01 level. B-ELISA detected a more than 3-fold rise in antibody titres in paired serum samples collected from mares aborting owing to EHV-1 infection. Mab 9C6 was chosen for testing 231 field sera from apparently healthy vaccinated and non-vaccinated horses from organized breeding farms belonging to 11 Indian states, and from Bhutan, by B-ELISA and VNT. There was very good agreement between the results obtained by both VNT and B-ELISA (K = 0.9438). Of 231 field sera, 144 samples were negative for EHV- 1 antibodies by both VNT and B-ELISA and 81 were positive by both tests. Two samples negative by VNT were found positive in B-ELISA. On the other hand, four weakly positive samples in VNT (VN antibody titre 0.9 1.2 log10) were negative in B-ELISA. The Mab (9C6)-based B-ELISA was found to be a suitable alternative to VNT for screening large numbers of field sera and enabled confirmatory EHV-1 serodiagnosis.

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.

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.