First isolation of an H1N1 avian influenza virus from wild terrestrial non-migratory birds in Argentina

A type A avian influenza (AI) virus was isolated from dead or severely ill red-winged tinamous (Rhynchotus rufescens) found in a hunting ground in April 2008 in Argentina. The subtype of A/red-winged tinamou/Argentina/MP1/2008 was determined as H1N1 by sequence analysis. The cleavage site of the viral hemagglutinin corresponded to a low pathogenic influenza virus, although the clinical presentation and pathological studies suggest that the virus was pathogenic for red-winged tinamous. Phylogenetic analysis of the viral genome suggested that while the hemagglutinin and neuraminidase genes were related to AIV from North America, the internal genes were most closely related to other South American isolates. These findings support the postulated South American phylogenetic lineage for AIV PB2, PB1, PA, M and NS genes, and suggest that the evolutionary pathways of HA and NA genes involve exchanges between the Northern and Southern hemispheres.     

Prevalence of Campylobacter species in wild birds of South Korea

Campylobacter species cause human gastrointestinal infections worldwide. They commonly inhabit intestines of avian species including wild birds. They might play a role in the spread of infections to humans and other bird species. The prevalence of Campylobacter species in 2164 faecal samples of wild birds (representing 71 species and 28 families) captured across the Korean peninsula was evaluated in this study. The overall prevalence was 15.3% (332/2164). Bird species belonging to the family Charadriidae had the highest isolation rate (30.0%), followed by those belonging to the families Ardeidae (26.4%), Turdidae (21.9%), and Anatidae (15.3%). The prevalence of Campylobacter spp. differed significantly according to migratory habit. Stopover birds were the most commonly infected (19.0%), followed by winter migratory (16.7%) and summer migratory birds (12.3%). However, indigenous birds showed very low prevalence (2.7%). Antimicrobial susceptibility tests were performed for 213 isolates. Results showed that Campylobacter jejuni isolates (n?=?169) exhibited resistance to nalidixic acid (5.3%), ciprofloxacin (3.0%), and tetracycline (1.8%), while Campylobacter lari (n?=?1) displayed resistance to nalidixic acid and ciprofloxacin. However, all Campylobacter coli isolates (n?=?20) were susceptible to all antimicrobials tested. This is the first report on the prevalence of Campylobacter species in wild birds that seasonally or indigenously inhabit the Korean peninsula. Our results indicate that the overall prevalence of Campylobacter in wild birds is moderate. Therefore, birds might serve as significant reservoirs for Campylobacter pathogens.     

Comparison of mice and cell cultures for the isolation of tick-borne viruses

Three methods of isolating viruses from 10 tick pools were compared; none of the methods produced all 13 of the viruses isolated (7 viruses of the bunyaviridae and 6 orbiviruses). Inoculation of homogenised ticks into various cell lines was the most successful, yielding 11 virus isolations. Only 2 tick homogenates induced overt signs of infection following intra-cerebral inoculation of 2-day-old mice. However, when inoculated mouse brain was passaged in various cell lines, 8 of 12 isolations were made. The rates of success of the 3 methods of virus isolation appeared to vary according to the type and titre of the virus in the tick pool.     

Use of the human colorectal adenocarcinoma (Caco‐2) cell line for isolating respiratory viruses from nasopharyngeal aspirates

The human colorectal adenocarcinoma‐derived Caco‐2 cell line was evaluated as a means isolating common respiratory viruses from nasopharyngeal aspirates for the diagnosis of respiratory diseases. One hundred eighty‐nine direct immunofluorescence positive nasopharyngeal aspirates obtained from patients with various viral respiratory diseases were cultured in the presence of Caco‐2 cells or the following conventional cell lines: LLC‐MK2, MDCK, HEp‐2, and A549. Caco‐2 cell cultures effectively propagated the majority (84%) of the viruses present in nasopharyngeal aspirate samples compared with any positive cultures obtained using the panel cells (78%) or individual cell line MDCK (38%), HEp‐2 (21%), LLC‐MK2 (27%), or A549 (37%) cell lines. The differences against individual cell line were statistically significant (P = < 0.000001). Culture in Caco‐2 cells resulted in the isolation of 85% (36/42) of viruses which were not cultivated in conventional cell lines. By contrast, 80% (24/30) of viruses not cultivated in Caco‐2 cells were isolated using the conventional panel. The findings indicated that Caco‐2 cells were sensitive to a wide range of viruses and can be used to culture a broad range of respiratory viruses. J. Med. Virol. 85:874–879, 2013. © 2013 Wiley Periodicals, Inc.     

Frequent inter-species transmission and geographic subdivision in avian influenza viruses from wild birds

Revealing the factors that shape the genetic structure of avian influenza viruses (AIVs) in wild bird populations is essential to understanding their evolution. However, the relationship between epidemiological dynamics and patterns of genetic diversity in AIV is not well understood, especially at the continental scale. To address this question, we undertook a phylogeographic analysis of complete genome sequences of AIV sampled from wild birds in North America. In particular, we asked whether host species, geographic location or sampling time played the major role in shaping patterns of viral genetic diversity. Strikingly, our analysis revealed no strong species effect, yet a significant viral clustering by time and place of sampling, as well as the circulation of multiple viral lineages in single locations. These results suggest that AIVs can readily infect many of the bird species that share breeding/feeding areas.     

Evaluation of Aedes albopictus tissue culture for use in association with arbovirus isolation.

The susceptibility and sensitivity of Aedes albopictus cell cultures to five different primary and four different low-passage arboviruses were tested. Yellow fever, West Nile, Ilesha, eastern equine encephalitis, and Flanders viruses replicated in A albopictus tissue cultures. Replication was determined by the ability of selected tissue culture fluids to infect suckling mice, and by recovery from tissue culture fluid of progressively increasing amounts of complement-fixing (CF) antigen with time. Virus persistence was demonstrated with Nodamura, western equine encephalitis, and Mayaro viruses, but multiplication was not proven; neither persistence nor multiplication was demonstrated with a Kemerovo group virus. When yellow fever and Ilesha viruses were simultaneously inoculated into A albopictus culture, CF antigen for each was consistently detected. In a more detailed comparative study of field specimens, 12 unpassaged strains of yellow fever virus were tested for infectivity in A albopictus tissue culture, Vero cells, and baby mice. Higher titers of virus were detected (0.8--2.3 log ID50 per ml) in Vero cell culture than in A albopictus tissue culture or baby mouse systems. These results suggest the feasibility of using A albopictus cells in association with the primary isolation of arboviruses.     

Phylogenic analysis of the M genes of influenza viruses isolated from free-flying water birds from their Northern Territory to Hokkaido, Japan

During 2000-2007, 218 influenza viruses of 28 different combinations of HA (H1-H13) and NA (N1-N9) subtypes were isolated from fecal samples of free-flying water birds at two distant lakes in Hokkaido, Japan. Phylogenic analysis of the matrix (M) genes of 67 strains, selected on the basis of their subtype combinations, revealed that A/duck/Hokkaido/W95/2006 (H10N8) was a reassortant whose M gene belonged to North American non-gull-avian and the other seven genes to Eurasian non-gull-avian lineages. The M genes of other 65 strains belonged to Eurasian non-gull-avian and the one to Eurasian-gull lineages. The M genes of 65 strains were grouped into three different sublineages, indicating that influenza viruses circulating in different populations of free-flying water birds have evolved independently in nature.     

流感病毒MDCK细胞培养条件的优化

目的优化流感病毒MDCK细胞培养条件,提高流感病毒分离率和流感监测效果。方法使用MDCK细胞培养法,比较传统的与优化的实验条件对流感病毒分离率和对血凝试验滴度的影响。结果不同的优化实验条件可明显提高流感病毒的分离率和滴度。结论已筛选出流感病毒MDCK细胞培养法适宜的条件。     

A 3D digital reconstruction of the components of the gas exchange tissue of the lung of the muscovy duck, Cairina moschata

To elucidate the shape, size, and spatial arrangement and association of the terminal respiratory units of the avian lung, a three-dimensional (3D) computer-aided voxel reconstruction was generated from serial plastic sections of the lung of the adult muscovy duck, Cairina moschata. The air capillaries (ACs) are rather rotund structures that interconnect via short, narrow passageways, and the blood capillaries (BCs) comprise proliferative segments of rather uniform dimensions. The ACs and BCs anastomose profusely and closely intertwine with each other, forming a complex network. The two sets of respiratory units are, however, absolutely not mirror images of each other, as has been claimed by some investigators. Historically, the terms 'air capillaries' and 'blood capillaries' were derived from observations that the exchange tissue of the avian lung mainly consisted of a network of minuscule air- and vascular units. The entrenched notion that the ACs are straight (non-branching), blind-ending tubules that project outwards from the parabronchial lumen and that the BCs are direct tubules that run inwards parallel to and in contact with the ACs is overly simplistic, misleading and incorrect. The exact architectural properties of the respiratory units of the avian lung need to be documented and applied in formulating reliable physiological models. A few ostensibly isolated ACs were identified. The mechanism through which such units form and their functional significance, if any, are currently unclear.     

Assessment of different isolation procedures for blastomeres from two-cell mouse embryos

As an extension of in-vitro fertilization and embryo transfer,detection of genetic and metabolic defects prior to implantationmight be possible in the future. The objective of pre-implaiitationdiagnosis would be to sample a minimal amount of cellular materialof the conceptus for diagnosis prior to transfer. Differentprotocols for isolating individual blastomeres from two-cellmouse embryos were evaluated. Two-cell mouse embryos were collectedand the zona pdlucida was removed by enzyme treatment (pronase),by exposure to acid Tyrode (pH = 2.5) or by mechanical force(suction into a small pipette, removal with a microblade). Individualblastomeres were obtained by exposure to a chelating agent (EDTA-glycmemixture), to Ca²⁺-Mg²⁺-free PBS or after isolation by mechanicalforce (bisection with a microblade or suction in a small pipette).The isolated blastomeres were then cultured in vitro withoutzonae pellucidae. All isolation procedures had a negative impacton the growth patterns of the isolated blastomeres. Differentabnormalities could be observed at the blastocyst stage includingembryos lacking visible compaction features, embryos with doubleblastocoeJk cavities and embryos with no inner cell mass (trophoblasticvesicles).     

Genetic characterization of avian influenza viruses isolated from waterfowl in southern part of South Korea in 2006

Aquatic birds are a reservoir of all known influenza A viruses. Avian influenza viruses have played a major role in the creation of pandemic influenza viruses in humans. In this study, we genetically characterized genes of nine isolates from waterfowl in Eulsukdo, a congregating place for migratory birds on the flyway of migration from Siberia, which is located in the southern part of South Korea. Phylogenic analysis showed that HA and NA genes of isolates belonged to Eurasian lineage, and lineage analysis showed that NS, PB1, PA, NP, and M genes of isolates clustered with Eurasian lineage, and PB2 genes of isolates belonged to North American or Eurasian lineage. Results suggest that the interregional transmission of genes of avian influenza viruses may occur in the migratory birds.     

An improved method for the isolation and culture of rat epidermal stem cells

The management of burns and injuries using novel treatment strategies involving epidermal stem cells (ESC) requires a better understanding of the biology of these cells, in particular, their isolation and the maintenance of their unique characteristics in culture. The purpose of this study was to describe an improved method for isolating putative ESC from fetal rat skin and to maintain them long term in culture. Single ESC suspensions were obtained from fetal rat skin by enzyme digestion containing 0.5% neutral protease. The target cells were harvested by rapid adherence on type IV collagen plates and were cultured in complex DMEM. After primary isolation, cells were continuously cultured in K-serum free medium. After reaching 70-80% confluence, the cells were digested with 0.25% trypsin at 37°C for 5-10 minutes, and passaged at a ratio of 1:2. The cultured ESC showed good growth, resulting in cell viability of over 98%. Four days later, clones containing 100-200 cells were detected, showing cobblestone-like characteristics. The rapidly adherent cells were positive for keratin 15, 19 and P63. Eighty three percent of cells expressed β1 integrin. The growth-curve showed that the rapidly adherent cells were in the exponential growth phase. The protocol described in this paper provides a simplified and effective method to isolate and maintain long-term culture of epidermal stem cells from fetal rat skin. This method should be valuable for isolating and studying ESC from various transgenic rat lines that are currently available.     

Continuous cell lines from the Muscovy duck as potential replacement for primary cells in the production of avian vaccines

Veterinary vaccines contribute to food security, interrupt zoonotic transmissions, and help to maintain overall health in livestock. Although vaccines are usually cost-effective, their adoption depends on a multitude of factors. Because poultry vaccines are usually given to birds with a short life span, very low production cost per dose is one important challenge. Other hurdles are to ensure a consistent and reliable supply of very large number of doses, and to have flexible production processes to accommodate a range of different pathogens and dosage requirements. Most poultry vaccines are currently being produced on primary avian cells derived from chicken or waterfowl embryos. This production system is associated with high costs, logistic complexities, rigid intervals between harvest and production, and supply limitations. We investigated whether the continuous cell lines Cairina retina and CR.pIX may provide a substrate independent of primary cell cultures or embryonated eggs. Viruses examined for replication in these cell lines are strains associated with, or contained in vaccines against egg drop syndrome, Marek's disease, Newcastle disease, avian influenza, infectious bursal disease and Derzsy's disease. Each of the tested viruses required the development of unique conditions for replication that are described here and can be used to generate material for in vivo efficacy studies and to accelerate transfer of the processes to larger production volumes.     

Studies by electron microscopy on the assembly of duck hepatitis B virus in the liver

Liver specimens from 1-day-old ducklings infected in ovo with maternally transmitted duck hepatitis B virus (DHBV) were examined by electron microscopy. Complete and incomplete DHBV particles were located within hypertrophied cisternae of the endoplasmic reticulum of the hepatocytes. The complete viral particles found intracellularly have inner cores with a diameter ranging from 35 to 37.5 nm and an outer coat or envelope. The whole particle measures approximately 45-65 nm in diameter. Naked core particles were located in the nuclei, free in the cytoplasm, and also near or on the cisternal membrane of the endoplasmic reticulum on the cytoplasmic face. Duck hepatitis B virions appear to share morphological characteristics with the viral coat and core of human hepatitis B virus (HBV). Electron microscopy suggested that the core particles of DHBV migrate from the nucleus into the cytoplasm through the nuclear pores. The complete viral particles are probably formed by protrusion of the core particles through the endoplasmic reticulum and by simultaneous encapsulation with a coat derived from the endoplasmic reticulum.     

A comparison of PCR with virus isolation and direct antigen detection for diagnosis and typing of genital herpes

Patients attending the genitourinary medicine clinic at Watford General Hospital, UK, were examined for clinical signs of genital herpes infection. Genital swabs were taken from 194 patients (126 female, 68 male) who presented with genital ulceration or symptoms which were suggestive of genital herpes infection. Swabs from these patients were tested by three methods: (i) Detection of herpes simplex virus (HSV) antigen by direct HSV enzyme immunoassay (EIA), (ii) HSV isolation in Vero cell culture and (iii) HSV polymerase chain reaction (PCR). HSV was detected in 76 patients (39%) by EIA, in 93 (48%) by isolation in cell culture, and in 115 (59%) by PCR. Isolation by cell culture has been considered as the “gold standard” for the detection of HSV in genital lesions, but in this study HSV PCR was significantly more sensitive. Comparison of the three methods was as follows: Cell culture vs. PCR: Sensitivity 93/115 (80.9%), Specificity 79/79 (100%). HSV EIA vs. PCR: Sensitivity 75/115 (65.2%), Specificity 78/79 (98.7%). HSV EIA vs. Cell culture: Sensitivity 75/93 (80.7%), Specificity 100/101 (99%). EIA was less effective in detecting HSV among recurrent than among first episode infections, in comparison to culture or HSV PCR. This is the first comparison of HSV PCR with two other routine diagnostic methods for confirming genital herpes infection in a symptomatic population. The infecting HSV type was identified by restriction digestion of 108 HSV amplicons: HSV-1: 37/108 (34%), HSV-2: 71/108 (66%). In this population HSV-1 causes a significant proportion of genital herpes cases, and HSV-1 genital infection was detected in significantly more first episode infections (40.3%) than among recurrent infections (22.2%). J. Med. Virol. 55:177–183, 1998. © 1998 Wiley-Liss, Inc.     

An efficient in vivo method for the isolation of Puumala virus in Syrian hamsters and the characterization of the isolates from Russia

Puumala virus (PUUV) and other Arvicolinae-borne hantaviruses are difficult to cultivate in cell culture. To isolate these hantaviruses efficiently, hantavirus nucleocapsid protein (NP)-positive but seronegative wild rodents were selected by NP-detection ELISA. Three of 68 Myodes glareolus captured in Samara, Russia, were NP-positive and seronegative. Syrian hamsters were inoculated with lung homogenates from NP-positive rodents for virus propagation. Virus isolation in vitro was carried out by inoculation of lung homogenates of NP-positive hamsters to Vero E6 cell monolayers. Two PUUV strains (Samara49/CG/2005 and Samara94/CG/2005) from M. glareolus were isolated in Vero E6 cells. Nucleotide and amino acid sequence identities of the S segment of these isolates to those of PUUV F-s808 from a fatal HFRS patient in Samara region were 96.7-99.3% and 99.3-100.0%, respectively. Morphologic features of Vero E6 cells infected with PUUV strain Samara49/CG/2005 were quite similar to those of Hantaan virus-infected cells. Isolation of Hokkaido virus from Myodes rufocanus captured in Hokkaido, Japan, was also performed. Hokkaido virus NP and RNA were recovered and maintained in hamsters. These results suggest that inoculation of Syrian hamsters with rodent samples is an efficient method for the isolation and maintenance of PUUV and other Arvicolinae-borne hantaviruses.     

一种分离、培养扩增小鼠NK细胞方法的建立

目的:建立小鼠NK细胞的分离及培养扩增的方法。方法:用两步黏附分离法和磁珠活化的细胞分选(MACS)法,从小鼠脾脏的单个核细胞(MNC)中分离NK细胞,计数所得细胞的总数,并用FITC抗小鼠CD3和PE抗小鼠NK1.1单克隆荧光抗体染色后,用流式细胞仪(FACS)检测NK细胞的纯度。以YAC -1为靶细胞,用MTT比色法检测NK细胞的杀伤活性。结果:将两步黏附分离法分离的2×107个脾MNC培养5、10、15、20d,计数细胞的总数并检测CD3-NK1.1 细胞百分率,分别为0.5×107、1.4×107、2.6×107、3.0×107和18.36%、43.44%、55.68%、60.03%。效靶细胞25∶1时,NK细胞的杀伤率分别为54.38%、66.54%、79.38%和83.86%,明显高于脾MNC(41.93%)(P<0.01)。MACS法纯化前后细胞的总数和CD3-NK1.1 细胞的百分率,分别为1.0×108、1.5×106和2.54%、93.60%;但纯化后的细胞在体外培养20d时,只扩增到1.9×106个。结论:用两步黏附分离法分离的小鼠NK细胞,培养2～3wk可获得大量的NK细胞,纯度可达55%～60%;在效靶细胞为25∶1时,NK细胞对YAC1细胞的杀伤率约为80%。