Sunil K. Mor

Spring Viremia of Carp Virus in Minnehaha Creek, Minnesota

Spring viremia of carp virus (SVCV) causes a highly contagious and serious disease of freshwater cyprinid fishes, generating significant economic and ecological impacts throughout the world. The SVCV is therefore listed as a notifiable pathogen by the International Organization for Animal Health. In June 2011, a significant mortality event of wild common carp Cyprinus carpio occurred in Minnehaha Creek near its confluence with Mississippi River Pool 2 in Minneapolis, Minnesota. Clinical signs of moribund fish included hemorrhagic lesions in the skin, eyes, and internal adipose tissue. The SVCV was isolated from pooled kidney and spleen of the fish. Rhabdovirus particles were seen upon examination of infected cell culture fluid by electron microscopy. The virus was confirmed to be SVCV subtype Ia by reverse transcription PCR and sequencing. This is the first report of SVCV within the state of Minnesota and the ninth documented case in North America.

Detection and molecular characterization of Porcine astrovirus strains associated with swine diarrhea

Astrovirus has been reported to be associated with diarrhea in pigs. The current study was conducted for the detection and molecular characterization of astroviruses in diarrheic pigs submitted to the Veterinary Diagnostic Laboratory, University of Minnesota. Intestinal contents from 269 pigs were examined by reverse transcription polymerase chain reaction (RT-PCR), and 62% were found positive for astroviruses. Of the positive samples, 20% were positive for astrovirus alone while astrovirus with rotavirus was detected in 58% of the samples. The remaining 22% revealed the presence of astrovirus along with Porcine hemagglutinating encephalomyelitis virus, Transmissible gastroenteritis virus, or Porcine circovirus-2. Sequencing the capsid gene of 56 randomly selected samples confirmed them to be Porcine astrovirus type 4 (PAstV-4) with 58–100% nucleotide identity within these viruses. Phylogenetic analysis revealed 2 possible subgroups. The results indicate that PAstV is present on swine farms in the United States and that it may be associated with diarrhea either alone or in combination with other enteric viruses. Further studies are needed to determine strain diversity among porcine astroviruses so that appropriate control strategies can be devised and implemented.

Isolation and Molecular Characterization of a Novel Picornavirus from Baitfish in the USA

During both regulatory and routine surveillance sampling of baitfish from the states of Illinois, Minnesota, Montana, and Wisconsin, USA, isolates (n = 20) of a previously unknown picornavirus were obtained from kidney/spleen or entire viscera of fathead minnows (Pimephales promelas) and brassy minnows (Hybognathus hankinsoni). Following the appearance of a diffuse cytopathic effect, examination of cell culture supernatant by negative contrast electron microscopy revealed the presence of small, round virus particles (∼30–32 nm), with picornavirus-like morphology. Amplification and sequence analysis of viral RNA identified the agent as a novel member of the Picornaviridae family, tentatively named fathead minnow picornavirus (FHMPV). The full FHMPV genome consisted of 7834 nucleotides. Phylogenetic analysis based on 491 amino acid residues of the 3D gene showed 98.6% to 100% identity among the 20 isolates of FHMPV compared in this study while only 49.5% identity with its nearest neighbor, the bluegill picornavirus (BGPV) isolated from bluegill (Lepomis macrochirus). Based on complete polyprotein analysis, the FHMPV shared 58% (P1), 33% (P2) and 43% (P3) amino acid identities with BGPV and shared less than 40% amino acid identity with all other picornaviruses. Hence, we propose the creation of a new genus (Piscevirus) within the Picornaviridae family. The impact of FHMPV on the health of fish populations is unknown at present.

Isolation and Characterization of a Turkey Arthritis Reovirus

SUMMARY. During the spring and summer of 2011, the Minnesota Veterinary Diagnostic Laboratory at the University of Minnesota received 14 submissions of 15-to-18-week-old tom turkeys that were recumbent with wing tip bruises (“wing walkers”) and uni- or bilateral swelling of the hock (tibiotarsal) joints. Gastrocnemius or digital flexor tendons were occasionally ruptured. A total of five turkey arthritis reoviruses (TARV-MN1 through TARV-MN5) were isolated in specific-pathogen-free embryonated chicken eggs and QT-35 cells. The identity of the isolates was confirmed by electron microscopy, reverse transcription-polymerase chain reaction, and gene sequence analysis. BLAST analysis on the basis of a 880 bp nucleotide sequence of the S4 gene confirmed all isolates as a reovirus. Phylogenetic analysis divided the five isolates into two subgroups: subgroup I containing TARV-MN1, -2, -3, and -5, and the other subgroup containing TARV-MN4. Isolates in subgroup I had a similarity of 97%–100% with each other, while subgroup II (TARV-MN4) had a similarity of only 89.2% with subgroup I viruses. This isolate showed 90%–93% similarity with turkey enteric reoviruses in the United States, while the other four isolates in subgroup I had 89%–97.6% similarity. These results indicate divergence within TARVs as well as from enteric viruses, which needs to be confirmed by complete genome sequence analysis. Further experimental studies are planned to determine the role of these isolates in turkey arthritis and to compare them with classical chicken reovirus. RESUMEN. Aislamiento y caracterización de un reovirus asociado con artritis en pavos. Durante la primavera y el verano del año 2011, el Laboratorio de Diagnóstico Veterinario el Estado de Minnesota en la Universidad de Minnesota recibió 14 casos de pavos machos de 15 a 18 semanas de edad, que mostraron recumbencia con moretones en la punta del ala (“aves que caminan con las alas”) e inflamación de la articulación del corvejón (articulación tibiotarsal) uni o bilateral. Ocasionalmente, los tendones del músculo gastrocnemio o flexores digitales se observaron rotos. Un total de cinco reovirus asociados con artritis en pavos (denominados TARV-MN1 al TARV-MN5) se aislaron en huevos embrionados de pollo libres de patógenos específico y en células QT-35. La identidad de los aislados se confirmó mediante microscopía electrónica, por transcripción reversa y reacción en cadena de la polimerasa, y por el análisis de secuencias. Mediante el análisis con la base de datos BLAST de las secuencias de nucleótidos de productos de 880 pares de bases que incluían al gene S4 confirmaron a todos los aislamientos como reovirus. El análisis filogenético dividió a los cinco aislamientos en dos subgrupos: subgrupo I que contenía a los virus TARV-MN1, TARV-MN2, TARV-MN3 y TARV-MN5 y el otro subgrupo que incluía al virus TARV-MN4. Los aislados en el subgrupo I mostraron una similitud de 97% -100% entre ellos, mientras que el subgrupo II (TARV-MN4) mostró una similitud de sólo 89.2% con los virus de subgrupo I. Este aislamiento mostró una similitud de 90%–93% con reovirus de pavo entéricos en los Estados Unidos, mientras que otros cuatro aislados en el subgrupo I mostraon una similitud de 89%–97.6%. Estos resultados indican divergencia dentro de los reovirus asociados con artritis en pavos, así como de los virus entéricos, lo cual debe ser confirmado por el análisis de las secuencias completas del genoma. Se han planeado otros estudios experimentales para determinar el papel de estos aislamientos en la artritis de los pavos y compararlas con reovirus de pollo clásicos.

The occurrence of enteric viruses in Light Turkey Syndrome

Two studies were conducted to determine the role of enteric viruses in Light Turkey Syndrome (LTS), which is characterized by lower weight in market age turkeys than their standard breed character. In the surveillance study, we selected four LTS and two non-LTS turkey flocks in Minnesota and collected faecal samples at 2, 3, 5 and 8-weeks of age. Astrovirus, rotavirus, and reovirus were detected alone or in various combinations in both LTS and non-LTS flocks. No coronavirus was detected in LTS flocks and no corona- or reovirus was detected in non-LTS flocks. In the second study, 2-week-old turkey poults were divided into two groups; Group A (challenged) was inoculated orally with 10% pooled faecal suspension from LTS flocks and group B (control) was inoculated with phosphate buffered saline (PBS). Clinical signs of depression, huddling, and lack of uniform size were observed in the challenged group but not in the control group. diarrhoea was observed in both groups but was more severe in the challenged group than in the control group. Birds in the challenged group shed astrovirus, rotavirus and reovirus, while the control group shed only astrovirus. Virus shedding in both groups was observed for up to nine weeks of age. Significantly lower body weights were seen in the challenged group starting at seven weeks of age and lasting until 20 weeks of age. These findings suggest that viral enteritis at an early age may set up conditions for the development of LTS in adult turkeys.

A Multiplex RT-PCR for the Detection of Astrovirus, Rotavirus, and Reovirus in Turkeys

SUMMARY. This study was undertaken to develop and validate a multiplex reverse transcription-polymerase chain reaction (mRT-PCR) for simultaneous detection of avian rotavirus, turkey astrovirus-2 (TAstV-2), and avian reovirus. Primers targeting the conserved regions of NSP4 gene of avian rotavirus, polymerase gene of TAstV-2, and S4 gene of avian reovirus were used. The position of bands at 630, 802, and 1120 base pairs on agarose gel confirmed the presence of rotavirus, TAstV-2, and reovirus, respectively. This mRT-PCR was found to be specific as no amplification was observed with avian influenza virus, Newcastle disease virus, turkey coronavirus, avian metapneumovirus, and intestinal contents of uninfected turkey poults. Intestinal contents of poults from flocks suspected of exhibiting “poult enteritis syndrome” were pooled and tested. Of the 120 pooled samples tested, 70% were positive for TAstV-2, 45% for avian rotavirus, and 18% for avian reovirus. These three viruses were detected alone or in different combinations. Of the samples tested, 20% were negative for these three viruses, 38% were positive for a single virus (TAstV or rotavirus or reovirus), and 42% were positive for two or three viruses. This single-tube mRT-PCR assay has the potential to serve as a rapid diagnostic method for the simultaneous detection of the three enteric viruses in turkeys.

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.

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

ABSTRACT 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.

Evolutionary, epidemiological, demographical, and geographical dissection of porcine bocavirus in China and America

Porcine bocavirus was first discovered in Swedish pigs with post-weaning multisystemic wasting syndrome (PMWS) in 2009. Many efforts have been implemented to investigate the porcine bocavirus, but it remains enigmatic. In the current study, we utilized data from both China and the USA. The China-derived data included 403 pig samples collected from five provinces, 122 gene sequences from the GenBank database, and 637 old porcine bocavirus (PBoV) cases. The USA-derived data comprised 181 pig samples from 18 states, 39 new gene sequences, and 85 new emerging cases. First, we executed a comprehensive analysis of the diseases prevalence, phylogenetics, evolutionary distances, mutation network, geographical distribution, occurrence frequency, and phylogeographical estimation in both China and the USA. The results showed that the positive rates of PBoV (42.0%, 76/181) in American samples were significantly higher than those (11.4%, 46/403) in the Chinese samples. All PBoV cases from these countries can be divided into six groups: PBoV1 (group 1), PBoV2 (group 2), PBoV3C (group 3), PBoV5 (group 4), PBoV3/4 (group 5), and PBoV6V7V (group 6). PBoV1 and PBoV2 were epidemic strains from 2006 to 2011 in China, whereas the PBoV3 subtypes were epidemic from 2010 to 2012 in China and the USA. At present, PBoV3C (group 3), PBoV5 (group 4), and PBoV3/4 (group 5) are epidemic viruses and co-exist in China and the USA. The geographical distribution of PBoV mainly lies in the east and south coastal areas of China and the central states of the USA. Jiangsu Province and the state of Minnesota were the centers of high occurrence frequency of PBoV with six outbreaks. The old PBoV cases involved 14 provinces and regions of China and North Carolina in the USA, whereas the new emerging cases involved five provinces in China and 13 states in the USA, of which two provinces and 12 states reported for the first time that piglets were infected by PBoV. Hong Kong, Hebei, and Jiangsu Provinces and the states of Minnesota and North Carolina were possibly geographical origins of PBoV in China and America, respectively. These data can help us systematically understand porcine bocavirus in China and America and find effective strategies for its treatment.