Harsha Verma

Association of Airborne Virus Infectivity and Survivability with its Carrier Particle Size

Although laboratory generated virus aerosols have been widely studied in terms of infectivity and survivability, how they are related to particle size, especially in the submicron size range, is little understood. Four viruses (MS2 bacteriophage, transmissible gastroenteritis virus, swine influenza virus, and avian influenza virus) were aerosolized, size classified (100–450 nm) using a differential mobility analyzer (DMA), and collected onto gelatin filters. Uranine dye was also nebulized with the virus, serving as a particle tracer. Virus infectivity assay and quantitative reverse transcription-polymerase chain reaction were then used to quantify the amount of infectious virus and total virus present in the samples, respectively. The virus distribution was found to be better represented by the particle volume distribution rather than the particle number distribution. The capacity for a particle to carry virus increased with the particle size and the relationship could be described by a power law. Virus survivability was dependent on virus type and particle size. Survivability of the three animal viruses at large particle size (300–450 nm) was significantly higher than at particle size close to the size of the virion (100–200 nm), which could be due to the shielding effect. The data suggest that particle size plays an important role in infectivity and survivability of airborne viruses and may, therefore, have an impact on the airborne transmission of viral illness and disease. The data in this study do not support the use of MS2 bacteriophage as a general surrogate for animal and human viruses. Copyright 2013 American Association for Aerosol Research

Survival of Airborne MS2 Bacteriophage Generated from Human Saliva, Artificial Saliva, and Cell Culture Medium

ABSTRACT Laboratory studies of virus aerosols have been criticized for generating airborne viruses from artificial nebulizer suspensions (e.g., cell culture media), which do not mimic the natural release of viruses (e.g., from human saliva). The objectives of this study were to determine the effect of human saliva on the infectivity and survival of airborne virus and to compare it with those of artificial saliva and cell culture medium. A stock of MS2 bacteriophage was diluted in one of three nebulizer suspensions, aerosolized, size selected (100 to 450 nm) using a differential mobility analyzer, and collected onto gelatin filters. Uranine was used as a particle tracer. The resulting particle size distribution was measured using a scanning mobility particle sizer. The amounts of infectious virus, total virus, and fluorescence in the collected samples were determined by infectivity assays, quantitative reverse transcription-PCR (RT-PCR), and spectrofluorometry, respectively. For all nebulizer suspensions, the virus content generally followed a particle volume distribution rather than a number distribution. The survival of airborne MS2 was independent of particle size but was strongly affected by the type of nebulizer suspension. Human saliva was found to be much less protective than cell culture medium (i.e., 3% tryptic soy broth) and artificial saliva. These results indicate the need for caution when extrapolating laboratory results, which often use artificial nebulizer suspensions. To better assess the risk of airborne transmission of viral diseases in real-life situations, the use of natural suspensions such as saliva or respiratory mucus is recommended.

Prevalence and complete genome characterization of turkey picobirnaviruses

Abstract The “light turkey syndrome” (LTS), in which birds weigh less than their standard breed character at the marketing time, is believed to be a consequence of viral enteritis at an early age (3–5weeks) from which the birds never fully recover. In a previously published study, we collected fecal pools from 2, 3, 5 and 8week old turkey poults (80 pools from LTS farms and 40 from non-LTS farms) and examined them for the presence of astro-, rota-, reo-, and coronaviruses. To determine the presence of additional enteric viruses, we analyzed a fecal pool by Illumina sequencing and found picobirnavirus (PBV). Segments 1 and 2 of this virus shared 45.8%aa and 60.9–64.5%aa identity with genogroup I of human PBV, respectively. Primers based on RNA-dependent RNA polymerase and capsid genes were designed for detection and molecular characterization of PBVs in the 120 fecal pools described above. From LTS farms, 39 of 80 (48.8%) pools were PBV positive while 23 of 40 (57.5%) were positive from non-LTS farms. The phylogenetic analysis of 15 randomly selected strains divided them into four subgroups within genogroup I (subgroups 1A–D). Nine strains were in subgroup IA showing 69.9–76.4%nt identity with human PBV GI strainVS111 from the Netherlands. Strains in subgroup IB (n =2) had 91.4–91.7%nt identity with chicken PBV GI strain AVE 42v1 from Brazil. Two strains in subgroup IC had 72.3–74.2%nt identity with chicken PBV strain AVE 71v3 from Brazil. In subgroup ID, two strains showed 72.4–81.8%nt identity with chicken PBV GI strain AVE 57v2 from Brazil. Subgroup IC and ID were the most divergent. Five of the 15 strains were typed using capsid gene primers. They showed 32.6–33.4%nt and 39.5–41.3%aa identity with VS10 human PBV strain. These results indicate co-circulation of divergent strains of PBVs among Minnesota turkeys.

Characterization of S class gene segments of a newly isolated turkey arthritis reovirus

We report on the complete characterization of S class gene segments of 12 newly isolated turkey arthritis reoviruses (TARVs) and compare it with that of a turkey enteric reovirus (TERV). Phylogenetic analysis of S2, S3 and S4 genome segments revealed grouping of all TARVs into two lineages while, on the basis of S1 genome segment, only one lineage was found. All TARVs had 95-100% nucleotide identity based on sigma C protein sequences (S1 segment) but varied from 90-100%, 88.9-100% and 88.7-100% on the basis of S2, S3, and S4 genome segments, respectively. Point mutations as well as possible re-assortments were observed in TARVs throughout the S class indicating the need for extensive epidemiological studies on these viruses in hatcheries and commercial farms, which would be useful in determining virus variation in the field.

Comparison of Thermal and Non-Thermal Processing of Swine Feed and the Use of Selected Feed Additives on Inactivation of Porcine Epidemic Diarrhea Virus (PEDV)

Infection with porcine epidemic diarrhea virus (PEDV) causes diarrhea, vomiting, and high mortality in suckling pigs. Contaminated feed has been suggested as a vehicle of transmission for PEDV. The objective of this study was to compare thermal and electron beam processing, and the inclusion of feed additives on the inactivation of PEDV in feed. Feed samples were spiked with PEDV and then heated to 120–145°C for up to 30 min or irradiated at 0–50 kGy. Another set of feed samples spiked with PEDV and mixed with Ultracid P (Nutriad), Activate DA (Novus International), KEM-GEST (Kemin Agrifood), Acid Booster (Agri-Nutrition), sugar or salt was incubated at room temperature (~25°C) for up to 21 days. At the end of incubation, the virus titers were determined by inoculation of Vero-81 cells and the virus inactivation kinetics were modeled using the Weibull distribution model. The Weibull kinetic parameter delta represented the time or eBeam dose required to reduce virus concentration by 1 log. For thermal processing, delta values ranged from 16.52 min at 120°C to 1.30 min at 145°C. For eBeam processing, a target dose of 50 kGy reduced PEDV concentration by 3 log. All additives tested were effective in reducing the survival of PEDV when compared with the control sample (delta = 17.23 days). Activate DA (0.81) and KEM-GEST (3.28) produced the fastest inactivation. In conclusion, heating swine feed at temperatures over 130°C or eBeam processing of feed with a dose over 50 kGy are effective processing steps to reduce PEDV survival. Additionally, the inclusion of selected additives can decrease PEDV survivability.

Experimentally induced lameness in turkeys inoculated with a newly emergent turkey reovirus.

Newly emergent turkey arthritis reoviruses (TARVs) have been isolated from cases of lameness in male turkeys over 10 weeks of age. In a previous study, experimental inoculation of TARV in one-week-old turkey poults produced lymphocytic tenosynovitis at four weeks post inoculation but without causing clinical lameness. This study was undertaken to determine if TARV infection at an early age can lead to clinical lameness in birds as they age. One-week-old male turkeys were inoculated orally with a TARV (strain TARV-O’Neil) and monitored for the development of gait defects until 16 weeks of age. At 4, 8, 12 and 16 weeks of age, a subset of birds was euthanized followed by the collection of gastrocnemius tendon, digital flexor tendon, and intestines for virus detection by rRT-PCR and for histologic inflammation scoring. Clinical lameness was first displayed in TARV-infected turkeys at 8 weeks of age and ruptured gastrocnemius tendons with progressive lameness were also seen at 12–16 weeks of age. The virus was detected in gastrocnemius tendon of 4- 8- and 12-week-old turkeys but not in 16-week-old turkeys. Histologic inflammation scores of tendons at each of the four time points were significantly higher in the virus-inoculated group than in the control group (p < 0.01). Lesions began as lymphocytic tenosynovitis with mild synoviocyte hyperplasia at four weeks of age and progressed to fibrosis as the birds aged. These results demonstrate the potential of TARV to infect young turkeys and to produce subclinical tenosynovitis that becomes clinically demonstrable as the turkeys age.

One-Step Real-Time Reverse Transcription–PCR for the Detection of Turkey Reoviruses

SUMMARY During late 2010 and early 2011, an unusual problem of lameness and swollen hock joints in commercial turkeys was reported in the upper Midwest, which continues to this day. The disease caused substantial economic losses to turkey producers. Reovirus was isolated from tendons and joint fluids of lame turkeys submitted to the Minnesota Veterinary Diagnostic Laboratory. This study was undertaken to develop a TaqMan real-time reverse transcription-PCR (rRT-PCR) assay for the early detection of turkey reoviruses (both enteric and lameness strains). A primer probe set was designed from the conserved region of the S4 segment of the turkey reovirus genome. The newly developed rRT-PCR was specific for the detection of turkey reoviruses. The detection limit of this assay was 10 genome copies per reaction. For the TARV-MN4 strain of turkey arthritis reovirus, one 50% tissue culture infectious dose was equivalent to 11.6 ± 0.2 genome copies. The highest coefficient of variation for intraexperimental and interexperimental variability was 0.08 and 0.06, respectively, indicating the reproducibility of the assay. This new test should be useful for the detection of turkey enteric and arthritis reoviruses. RESUMEN Método de transcripción reversa y PCR en un solo paso para la detección de reovirus de pavos. Durante finales del año 2010 y principios del 2011, se informó de un problema poco común de cojera e inflamación de corvejones en pavos comerciales en el Medio Oeste de los Estados Unidos, que continúa hasta este momento. La enfermedad ha causado substanciales pérdidas económicas a los productores de pavo. Se aisló un reovirus de los tendones y de los fluidos de las articulaciones de los pavos con cojera enviados al Laboratorio de Diagnóstico Veterinario de Minnesota. Este estudio se realizó para desarrollar un método de transcripción reversa y PCR en tiempo real (rRT-PCR) con sondas TaqMan para la detección temprana de los reovirus de pavo (cepas entéricas y cepas causantes de cojera). Se diseñó un conjunto de iniciadores para la región conservada del segmento S4 del genoma del reovirus de pavo. El método de rRT-PCR recientemente desarrollado era específico para la detección de reovirus de pavo. El límite de detección de este ensayo fue de 10 copias de genoma por reacción. Para la cepa TARV-MN4 de reovirus de pavo causante de artritis, una doses infectante 50% en cultivo de tejidos fue equivalente a 11.6 ± 0,2 copias del genoma. Los mayores coeficientes de variación para la variabilidad intraexperimental e interexperimental fueron de 0.08 y 0.06, respectivamente, indicando la reproducibilidad del ensayo. Esta nueva prueba puede ser útil para la detección de reovirus de pavos entéricos y causantes de artritis.

Environmental persistence of porcine coronaviruses in feed and feed ingredients

Porcine Epidemic Diarrhea Virus (PEDV), Porcine Delta Corona Virus (PDCoV), and Transmissible Gastroenteritis Virus (TGEV) are major threats to swine health and contaminated feed plays a role in virus transmission. The objective of our study was to characterize inactivation of PEDV, PDCoV, and TGEV in various feed ingredient matrices. Samples of complete feed, spray dried porcine plasma, meat meal, meat and bone meal, blood meal, corn, soybean meal, and corn dried distillers grains with solubles were weighed (5 g/sample) into scintillation vials and inoculated with 1 mL of PEDV, PDCoV, or TGEV. Samples were incubated at room temperature for up to 56 days. Aliquots were removed at various time points followed by preparing serial 10-fold dilutions and inoculating in cell cultures to determine the amount of surviving virus. Inactivation kinetics were determined using the Weibull model, which estimates a delta value indicating the time necessary to reduce virus concentration by 1 log. Delta values of various ingredients were compared and analyzed as to their nutrient composition. Soybean meal had the greatest delta value (7.50 days) for PEDV (P < 0.06) as compared with all other ingredients. High delta values (P < 0.001) were observed in soybean meal for PDCoV (42.04 days) and TGEV (42.00 days). There was a moderate correlation between moisture content and the delta value for PDCoV (r = 0.49, P = 0.01) and TGEV (r = 0.41, P = 0.02). There was also a moderate negative correlation between TGEV survival and ether extract content (r = -0.51, P = 0.01). In conclusion, these results indicate that the first log reduction of PDCoV and TGEV takes the greatest amount of time in soybean meal. In addition to this, moisture and ether content appear to be an important determinant of virus survival in feed ingredients.

0176 Environmental persistence of porcine epidemic diarrhea virus, porcine delta corona virus, and transmissible gastroenteritis in feed ingredients

Abstract Porcine epidemic diarrhea virus (PEDV), porcine delta corona virus (PDCoV), and transmissible gastroenteritis (TGEV) are major threats to swine production. Investigations of recent outbreaks confirmed that contaminated feed plays a role in virus transmission. This risk makes it necessary to evaluate the survival of such viruses in various feed ingredients. The objective of our experiment was to characterize the inactivation of PEDV, PDCoV, and TGEV in various feed and ingredient matrices. To determine differences in virus survival, 5-g samples of complete feed, spray-dried porcine plasma, meat meal, meat and bone meal, blood meal, corn, soybean meal, and low, medium, and high oil dried distillers grains with solubles were weighed into separate scintillation vials. These samples were inoculated with 1 mL of PEDV, PDCoV, or TGEV and incubated at room temperature for up to 56 d. At each time point, surviving virus was eluted and the supernatant was inoculated into vero-81 cells for PEDV, or swine testicular cells for PDCoV and TGEV. Cells were observed daily for 10 d for cytopathic effects, and this information was used to calculate a median tissue culture infectious dose (TCID50) using the Karber method. Inactivation kinetics were determined using the Weibull model. A delta value was estimated from the model, indicating the time necessary to reduce virus concentration by 1 log. This delta value was then compared across ingredients using the mixed procedure of SAS, and correlations between ingredient proximate analysis data and delta values were determined. Results showed that soybean meal had the greatest delta value (7.50 d) for PEDV compared with other ingredients (P < 0.06). Likewise, PDCoV (42.04 d) and TGEV (42.00 d) delta values were highest in soybean meal (P < 0.001). There was a moderate positive correlation between moisture and the delta value for PDCoV (r = 0.49, P = 0.01) and TGEV (r = 0.41, P = 0.02). There was also a moderate negative correlation between lipid content and the delta value for TGEV (r = −0.51, P = 0.01), suggesting that TGEV is less stable in ingredients with greater lipid content compared with ingredients with less lipid content. In conclusion, these results indicate that the first log reduction of PDCoV and TGEV takes the greatest amount of time in soybean meal and it appears to be the result of greater moisture content.

Phylogenetic analysis, genomic diversity and classification of M class gene segments of turkey reoviruses.

From 2011 to 2014, 13 turkey arthritis reoviruses (TARVs) were isolated from cases of swollen hock joints in 2-18-week-old turkeys. In addition, two isolates from similar cases of turkey arthritis were received from another laboratory. Eight turkey enteric reoviruses (TERVs) isolated from fecal samples of turkeys were also used for comparison. The aims of this study were to characterize turkey reovirus (TRV) based on complete M class genome segments and to determine genetic diversity within TARVs in comparison to TERVs and chicken reoviruses (CRVs). Nucleotide (nt) cut off values of 84%, 83% and 85% for the M1, M2 and M3 gene segments were proposed and used for genotype classification, generating 5, 7, and 3 genotypes, respectively. Using these nt cut off values, we propose M class genotype constellations (GCs) for avian reoviruses. Of the seven GCs, GC1 and GC3 were shared between the TARVs and TERVs, indicating possible reassortment between turkey and chicken reoviruses. The TARVs and TERVs were divided into three GCs, and GC2 was unique to TARVs and TERVs. The proposed new GC approach should be useful in identifying reassortant viruses, which may ultimately be used in the design of a universal vaccine against both chicken and turkey reoviruses.