Aplicação da técnica de RT-PCR para metapneumovírus aviário (aMPV) / Application of RT-PCR technique for avian metapneumovirus (aMPV) / Aplicación de la técnica RT-PCR para metapneumovirus aviario (aMPV)

Os principais hospedeiros do Metapneumovírus aviário (aMPV) são os frangos de corte e perus. O vírus acomete o trato respiratório superior dos perus desencadeando a Rinotraqueíte Viral dos Perus (RVP). O principal objetivo deste trabalho foi padronizar uma técnica de RT-PCR para a detecção do aMPV, por meio do uso do kit AccessQuick™ RT-PCR system (Promega®). Foram utilizados amostras de suabes de traqueia e pulmão de 38 perus comerciais com sintomatologia respiratória e dois suabes oculares de faisão. O RNA viral foi extraído utilizando-se o kit RTP® DNA/RNA Virus Mini Kit (STRATEC Molecular). Em seguida as amostras foram submetidas à RT-PCR One Step, utilizando o kit AccessQuick™ RT-PCR system (Promega®). Todas as 40 amostras testadas por RT-PCR foram negativas, exceto a amostra vacinal que foi utilizada como controle positivo. O aMPV não causa latência em frangos de corte ou perus, logo a excreção viral é limitada. Dessa forma, a ausência da detecção de genoma viral neste estudo pode ser justificada devido à idade que as amostras foram coletadas em perus, com 140 dias no abatedouro, impossibilitando dessa maneira a amplificação do genoma do aMPV. Porém, esse estudo também mostra que a RT-PCR se mostrou eficaz para detectar o genoma viral do aMPV, podendo dessa forma ser utilizado como uma ferramenta de diagnóstico rápido para investigação e estudo de casos de aMPV em rebanho de perus.

The main hosts of Avian metapneumovirus (aMPV) are broilers and turkeys. This virus affects the upper respiratory tract of turkeys, triggering Turkey Rhinotracheitis (TRT). The aim of this study was to optimize a RT-PCR technique in order to detect aMPV using the AccessQuick™ RT-PCR system (Promega®) kit. Tracheal and lung swab samples from 38 commercial turkeys with respiratory symptoms and two ocular swabs from pheasants were analyzed. Viral RNA was extracted using RTP® DNA/RNA Virus Mini Kit (Molecular STRATEC) kit. All 40 samples tested were negative in the RT-PCR. The only positive sample was a vaccine strain, used as the positive control. The aMPV does not cause latency in broilers, chickens or turkeys, thus, the viral excretion is limited. However, the absence of viral genome detection in this study may be justified due to the age the samples were collected, since they were collected in turkeys with about 140 days in the slaughterhouse, thus preventing the amplification of the aMPV genome. This study shows that the RT-PCR is effective to detect aMPV viral genome and may be used as a rapid diagnostic tool for research and for the studying of aMPV cases in turkey flocks in Brazil.

Los principales anfitriones de Metapneumovirus aviario (aMPV) son los pollos de engorde y pavos. El virus afecta el tracto respiratorio superior de los pavos desencadenando la Rinotraqueitis Viral de los pavos (RVP). El principal objetivo de ese estudio fue estandarizar una técnica de RT-PCR para la detección del aMPV, a través del uso del kit AccessQuick™-PCRsystem (Promega®). Se utilizaron muestras de hisopos traqueales y pulmonares de 38 pavos comerciales con síntomas respiratorios y dos hisopos oculares de faisán. El RNA viral se extrajo utilizando el kit DNA RTP® DNA/RNA Virus Mini Kit (STRATEC Molecular). A continuación, las muestras se sometieron a la RT-PCR OneStep utilizando el kit AccessQuick™ RT-PCR (Promega®). Todas las 40 muestras analizadas por RT-PCR fueron negativas, excepto la muestra de vacuna que se utilizó como control positivo. El aMPV no causa latencia en pollos de engorde o pavos, por lo que la excreción viral es limitada. Así, la ausencia de la detección de genoma viral en este estudio puede ser justificada debido a la edad que se recogieron las muestras en los pavos, con 140 días en el matadero, imposibilitando de este modo la amplificación del genoma del aMPV. Sin embargo, ese estudio también muestra que la RT-PCR se ha demostrado eficaz para detectar el genoma viral del aMPV, pudiendo así ser utilizado como una herramienta de diagnóstico rápido para investigación y estudio de casos de aMPV en bandada de pavos.

Adenoviruses of canine and human origins in stool samples from free-living pampas foxes (Lycalopex gymnocercus) and crab-eating foxes (Cerdocyon thous) in São Francisco de Paula, Rio dos Sinos basin / Adenovírus de origens canina e humana em fezes de graxains (Lycalopex gymnocercus) e cachorros-do-mato (Cerdocyon thous) de vida livre em São Francisco de Paula, bacia do Rio dos Sinos

The spread of enteric viruses of domestic animals and human beings to wild species can be facilitated by the resistance of these viruses on the environment and their ability to be transmitted by water and contaminated food. The health status of the populations of pampas foxes (Lycalopex gymnocercus) and crab-eating foxes (Cerdocyon thous) is largely unknown and the landscapes occupied by these animals in southern Brazil have been threatened by human occupation and expansion of agriculture. In this work, the search of genomes of human and canine adenoviruses in feces from these wild carnivores was used to track the dissemination of domestic animals and human pathogens to the free-living populations in a wildlife reserve located in southern Brazil. This was performed by virus-specific differential real-time polymerase chain reactions (qPCR) on stool specimens, avoiding capture and additional stress to the animals. Genus-specific conventional reverse-transcriptase PCR (RT-PCR) was complementarily performed aiming the detection of enteroviruses (EV) and rotaviruses (RV) on these same samples. HAdV genomes were found on 14 out of the 17 (82.35%) stool samples analysed, whereas CAV was found co-infecting 5 of these samples. RV genomes were detected on 7 of the 17 samples (41.18%) and all samples were negative for EV. The results point to the dispersion of HAdV and RV at a high rate to these species of South American wild carnivores, which can be an effect of growing anthropisation of the habitat of these animals.

A disseminação de vírus entéricos de animais domésticos e seres humanos para espécies selvagens pode ser facilitada pela resistência desses vírus no ambiente e sua capacidade de ser transmitida por água e alimentos contaminados. O estado de saúde das populações de Graxains-do-campo (Lycalopex gymnocercus) e Cachorros-do-mato (Cerdocyon thous) é em grande parte desconhecida e as paisagens ocupadas por estes animais no sul do Brasil têm sido ameaçadas pela ocupação humana e a expansão da agricultura. Neste trabalho, utilizou-se a pesquisa de genomas de adenovírus humanos (HAdV ) e caninos (CAV-1 e -2) em amostras fezes desses carnívoros selvagens com vistas a diagnosticar a disseminação de patógenos de animais domésticos e seres humanos às populações de vida livre em uma reserva de vida selvagem, localizado no sul do Brasil. Foram realizadas reações em cadeia da polimerase diferenciais e em tempo real (qPCR) de adenovírus específicos em amostras de fezes, evitando a captura e estresse adicional para os animais. PCRs gênero-específicas convencionais com transcrição reversa prévia (RT-PCR) foram ainda realizadas visando a detecção de enterovírus (EV) e rotavírus (RV) nestas mesmas amostras. Genomas de HAdV foram encontrados em 14 a 17 amostras de fezes (82.35%) analisados, Considerando que o CAV foi encontrado coinfectando 5 destas amostras. Genomas de RV foram detectados em 7 das 17 amostras (41.18%) e todas as amostras foram negativas para EV. Os resultados apontam para a dispersão de HAdV e RV em uma taxa elevada para estas espécies de carnívoros selvagens sul-americanas, que podem ser um efeito da crescente antropização do habitat desses animais.

A reverse transcription loop-mediated isothermal amplification assay to rapidly diagnose foot-and-mouth disease virus C

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to rapidly detect foot-and-mouth disease virus serotype C (FMDV C). By testing 10-fold serial dilutions of FMDV C samples, sensitivity of the FMDV C RT-LAMP was found to be 10 times higher than that of conventional reverse transcription-PCR (RT-PCR). No cross-reactivity with A, Asia 1, or O FMDV or swine vesicular disease virus (SVDV) indicated that FMDV C RT-LAMP may be an exciting novel method for detecting FMDV C.

M gene analysis of canine coronavirus strains detected in Korea

The purpose of this study was to investigate the genetic features of canine coronavirus (CCV) strains detected in Korea. M gene sequences obtained for isolates from 22 dogs with enteritis over a 5-year period were evaluated. Sequence comparison revealed that the 22 Korean CCV strains had an 87.2 to 100% nucleotide homology. Comparing to the typical reference CCV strains (type II), the nucleotide sequence of Korean strains had homology ranged from 86.3% to 98.3% (89.1% to 99.2% for the amino acid sequence) and 87.7% to 97.8% (92.4% to 100% for the amino acid sequence) when compared to FCoV-like CCV strains (type I). Three amino acid variations in the M gene were characteristic for the Korean CCV strains. Phylogenetic analysis demonstrated that the 22 Korean CCV strains belonged to four typical CCV clusters (i.e., a unique Korean CCV cluster, a type II and transmissible gastroenteritis virus cluster, an intermediate cluster between type I and II, and a type I cluster). This study was the first to identify genetic differences of the M gene from Korean CCV strains and provided a platform for molecular identification of different Korean CCV strains.

Emergence of virulent pseudorabies virus infection in Northern China

Our investigation was conducted in order to verify a recent severe epidemic at several swine farms in northern China that indicated a newly emerging disease. Evidence confirmed that the epidemic was caused by a virulent Pseudorabies virus infection in swine herds.

Identification of differentially expressed genes in gauze-exposed omentum of dogs using differential display RT-PCR

Molecular mechanisms governing peritonitis caused by the presence of aseptic gauze have remained unclear. To identify the genes involved, sterile gauze-exposed omentum was collected at 0, 6, 12, 24, and 48 h intervals, and analyzed by differential display RT(reverse transcription)-PCR. Among over 1,200 bands, 230 bands were found differentially expressed. These bands represented the fragment sizes of approximately 200 to 1,500 bp. The eight fragments were expressed differentially in the treatment group but not in the control. The sequences of two bands were similar to those of genes associated with the inflammatory process and a band was related to repair and regeneration process. Another one was related with spermatogonia and the rest four were unknown. Additionally, amplicons corresponding to the full-length sequences of two inflammatory gene fragments were synthesized by rapid amplification of cDNA end PCR. One showed 99% similarity to the major histocompatibility complex class II dog leukocyte antigen-DR beta chain and the other was canis familiaris proteasome beta type 3. Results of the present study suggested that sterile gauze induced the differential expression of genes in the omentum involved in inflammation and healing process.

Prolonged excretion of a low-pathogenicity H5N2 avian influenza virus strain in the Pekin duck

H5N2 strains of low-pathogenicity avian influenza virus (LPAIV) have been circulating for at least 17 years in some Mexican chicken farms. We measured the rate and duration of viral excretion from Pekin ducks that were experimentally inoculated with an H5N2 LPAIV that causes death in embryonated chicken eggs (A/chicken/Mexico/2007). Leghorn chickens were used as susceptible host controls. The degree of viral excretion was evaluated with real-time reverse transcriptase-polymerase chain reaction (RRT-PCR) using samples from oropharyngeal and cloacal swabs. We observed prolonged excretion from both species of birds lasting for at least 21 days. Prolonged excretion of LPAIV A/chicken/Mexico/2007 is atypical.

Genetic diversity of porcine reproductive and respiratory syndrome virus in Korea

The high genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV) has been an obstacle to developing an effective vaccine for porcine reproductive and respiratory syndrome (PRRS). This study was performed to assess the degree of genetic diversity among PRRSVs from Korean pig farms where wasting and respiratory syndrome was observed from 2005 to 2009. Samples from 786 farms were tested for the presence of PRRSV using reverse transcription PCR protocol. A total of 117 farms were positive for type 1 PRRSV while 198 farms were positive for type 2. Nucleotide sequences encoding the open reading frame (ORF) 5 were analyzed and compared to those of various published PRRSV isolates obtained worldwide. Sequence identity of the ORF 5 in the isolates was 81.6~100% for type 1 viruses and 81.4~100% for type 2 viruses. Phylogenetic analysis of the ORF 5 sequences showed that types 1 and 2 PRRSVs from Korea were mainly classified into three and four clusters, respectively. The analyzed isolates were distributed throughout the clusters independent of the isolation year or geographical origin. In conclusion, our results indicated that the genetic diversity of PRRSVs from Korean pig farms is high and has been increasing over time.

Prevalence of hepatitis A virus in sea food in Iran / Prevalência de vírus da hepatite A em frutos do mar no Irã

The objective of this study was to determine the prevalence of Hepatitis A Virus (HAV) in sea food samples in the Isfahan and Shahrekord townships in Iran. From September 2010 to April 2011, a total of 300 samples of fresh fish, shrimp, crab and lobster were obtained from randomly selected retail stores in the Isfahan and Shahrekord townships in Iran. The samples were tested for the presence of HAV using a reverse transcriptase- polymerase chain reaction method. Out of the total number of samples examined, 8 (2.7%) were found to be positive for HAV. This virus was detected in 5% and 1.7% of fresh fish and shrimp, respectively. This study shows the importance of sea food as potential sources of HAV infection in people in Iran.

O objetivo deste estudo foi determinar a prevalência do vírus Hepatitis A (HAV) em amostras de frutos do mar nas cidades de Isfahan e Shahrekord no Iran. De setembro de 2010 a Abril de 2011 um total de 300 amostras de peixe fresco, camarão, caranguejo e lagosta foram obtidas de lojas de varejo aleatoriamente escolhidas nas cidades de Isfahan e Shahrekord no Iran. As amostras foram testadas para presença de HAV usando o método de reação em cadeia em transcriptase reversa. Do total de amostras examinadas, 8 (2.7%) foram positivas para HAV. Este vírus foi detectado em 5% e 1.7% de peixe fresco e camarão, respectivamente. Este estudo mostrou a importância de frutos do mar como fontes potenciais de infecção HAV em pessoas no Iran.

Molecular characterization of group A bovine rotavirus in southeastern and central-western Brazil, 2009-2010 / Caracterização molecular de rotavírus bovino do grupo A nas regiões Sudeste e Centro-Oeste do Brasil

Rotavirus is an important cause of neonatal diarrhea in humans and several animal species, including calves. A study was conducted to examine 792 fecal samples collected from calves among 65 dairy and beef herds distributed in two of Brazil's major livestock producing regions, aiming to detect the occurrence of rotavirus and perform a molecular characterization of the rotavirus according to G and P genotypes in these regions. A total of 40 (5.05%) samples tested positive for rotavirus by the polyacrylamide gel electrophoresis (PAGE) technique. The molecular characterization was performed by multiplex semi-nested RT-PCR reactions, which indicated that the associations of genotypes circulating in herds in Brazil's southeastern region were G6P[11], G10P[11], G[-]P[5] + [11], G[-]P[6] in the state of São Paulo and G6P[11], G8P[5], G11P[11], G10P[11] in the state of Minas Gerais. In the central-western region, the genotypes G6P[5] + [11], G6P[5], G8P[-], G6P[11], G [-] P[1], G[-] P[11], and G[-] P[5] were detected in the state of Goiás, while the genotypes G6P[5], G8[P11], G6[P11], G8[P1], G8[P5], G6[P1] were circulating in herds in the state of Mato Grosso do Sul. The genotypic diversity of bovine rotavirus found in each region under study underlines the importance of characterizing the circulating samples in order to devise the most effective prophylactic measures.

Rotavírus é uma importante causa de diarreia neonatal em humanos e várias espécies animais, incluindo bezerros. Foi realizado um estudo a partir de 792 amostras fecais colhidas de bezerros, provenientes de 65 rebanhos de leite e corte distribuídos em duas das maiores regiões produtoras no Brasil, com o objetivo de se detectar a ocorrência de rotavírus e realizar a sua caracterização molecular quanto aos genotipos G e P nestas regiões. Um total de 40 (5,05%) de amostras testadas foram positivas para rotavírus pela técnica de eletroforese em gel de poliacrilamida (PAGE). A caracterização molecular foi realizada através de reações do tipo Multiplex semi-nested RT-PCR demonstrando que as associações de genotipos circulantes em rebanhos da região Sudeste foram G6P[11], G10[P11], G[-]P[5]+[11], G[-]P[6] no Estado de São Paulo e G6P[11], G8P[5], G11P[11], G10P[11] no Estado de Minas Gerais. Na região Centro-Oeste, foram detectados no Estado de Goiás os genotipos G6P[5]+[11], G6P[5], G8P[-], G6P[11], G[-]P[1], G[-]P[11], G[-]P[5] enquanto os genotipos G6P[5], G8[P11], G6[P11], G8[P1], G8[P5], G6[P1] eram circulantes em rebanhos do Estado do Mato Grosso do Sul. A diversidade genotípica de rotavírus bovino encontrada em cada região estudada justifica a importância da caracterização das amostras circulantes para medidas profiláticas mais efetivas.

Detecção molecular e análise filogenética do gene H de amostras do vírus da cinomose canina em circulação no município de Campinas, São Paulo / Molecular detection and phylogenetic analysis of the gene H from canine distemper virus isolates circulating at the municipality of Campinas, São Paulo

O vírus da cinomose canina (CDV), um Morbillivirus da família Paramyxoviridae, é o agente etiológico de doença neurológica e sistêmica em cães. O diagnóstico laboratorial da infecção requer o isolamento viral ou detecção do material genético do vírus em secreções ou tecidos de cães com suspeita clínica da doença. A diversidade genética entre os isolados de CDV pode ser aferida pelo sequenciamento efilogenia molecular do gene que codifica a hemaglutinina viral (gene H), havendo atualmente um especial interesse em comparar as amostras circulantes a campo com o genogrupo América-1, que abrange as cepas presentes nas vacinas disponíveis no mercado. No presente estudo, foi realizada a detecção molecular do gene H de CDV a partir de amostras biológicas colhidas ante- e post- -mortem de 15 cães com sinais clínicos sugestivos de cinomose na região metropolitana de Campinas, São Paulo. Dez dos 15 cães analisados tiveram ao menos um órgão positivo na detecção molecular e os amplicons obtidos foram submetidos ao sequenciamento nucleotídico seguido de análise filogenética molecular. De forma semelhante ao que já foi reportado para estudo analisando a diversidade do gene H em outros países, a reconstrução filogenética obtida para as amostras de casos de cinomose da região de Campinas demonstrou as mesmas foram agrupadas junto a amostras norte-americanas, europeias e japonesas recentes, em um grupo genético distinto do grupo de amostras clássicas de CDV, nomeado America-1, o qual engloba as estirpes vacinais Snyder Hill, Onderstepoort e Lederle.

Canine distemper virus (CDV), a Morbillivirus of the family Paramyxoviridae, is the etiological agent of neurological and systemic disease in dogs. The laboratory diagnosis of infection requires viral isolation or detection of genetic material of the virus in secretions or tissues of dogs with clinical suspicion of the disease. The genetic diversity among isolates of CDV can be assessed by sequencing and phylogenetic analysis of the gene that encodes the viral hemagglutinin (H gene), and there is currently a special interest in comparing the strains currently circulating in the field with the genogroup America-1, which comprises strains present in vaccines available in the market. In this study, the molecular detection of CDV gene H was performed from biological samples harvested ante-and post-mortem from 15 dogs with clinical signs suggestive of canine distemper in the metropolitan region of Campinas, São Paulo. Ten of the 15 dogs examined had at least one positive organ under molecular detection and the obtained amplicons were sequenced and further analyzed by molecular phylogenetic analysis. Similarly to what has already been reported on previous studies regarding the diversity of the gene H in other countries, the phylogenetic reconstruction obtained for the samples of cases of distemper from Campinas region showed they were grouped with the North American, European and Japanese newly described samples, a genetic group distinguished from classical samples of CDV, named America-1, which encompasses the vaccine strains Snyder Hill, Onderstepoort and Lederle.

Peste des petits ruminants virus detected in tissues from an Asiatic lion (Panthera leo persica) belongs to Asian lineage IV

In this study, peste des petits ruminants virus (PPRV) was detected in frozen pooled tissue samples from a dead Asiatic lion (Panthera leo persica). The samples were negative for canine distemper virus and positive for PPRV nucleic acids when tested with one-step RT-PCR using the appropriate virus-specific primers. Subsequent amplification, cloning, and sequencing of the partial nucleocapsid, matrix, and fusion genes confirmed the presence of PPRV nucleic acid. Comparative sequence and phylogenetic analyses of the structural genes of the isolated virus confirmed that the virus belonged to Asian lineage IV and was closely related to PPRV circulating in India.

Frequency of group A rotavirus with mixed G and P genotypes in bovines: predominance of G3 genotype and its emergence in combination with G8/G10 types

The present study describes the genotypic distribution of rotaviruses (RVs) in an Indian bovine population with unexpectedly higher proportions of G3 alone or in combination of G8/G10. PCR-genotyping confirmed that 39.4% (13/33) of the prevalent RVs were the G3 type while 60.6% (20/33) were dual G3G10 or G3G8 types. P typing revealed that 93.9% (31/33) of the samples were P[11] while 6.1% (2/33) possessed a dual P[1]P[11] type. Sequence analysis of the VP7 gene from G3 strains viz. B-46, 0970, and BR-133 showed that these strains had sequence identities of 90.5% to 100% with other bovine G3 strains. The highest identity (98.9% to 100%) was observed with RUBV3 bovine G3 strains from eastern India. The G3 strains (B-46, 0970, and BR-133) showed 97.5% to 98.8% sequence homologies with the Indian equine RV strain Erv-80. Phylogenetic analysis demonstrated that G3 strains clustered with bovine RUBV3 and J-63, and equine Erv-80 G3. Overall, these results confirmed that the incidence of infection by RVs with the G3 genotype and mixed genotypes in the bovine population was higher than previously predicted. This finding reinforces the importance of constantly monitoring circulating viral strains with the G3 genotype in future surveillance studies.

Isolation and genetic characterization of Japanese encephalitis virus from equines in India

Japanese encephalitis (JE) is an important vector-borne viral disease of humans and horses in Asia. JE outbreaks occur regularly amongst humans in certain parts of India and sporadic cases occur among horses. In this study, JE seroprevalence and evidence of JE virus (JEV) infection among horses in Haryana (India) is described. Antibodies against JEV were detected in 67 out of 637 (10.5%) horses screened between 2006 and 2010. Two foals exhibiting neurological signs were positive for JEV RNA by RT-PCR; JEV was isolated from the serum of one of the foals collected on the second day of illness. This is the first report of JEV isolation from a horse in India. Furthermore, a pool of mosquitoes collected from the premises housing these foals was positive for JEV RNA by RT-PCR. Three structural genes, capsid (C), premembrane (prM), and envelope (E) of the isolated virus (JE/eq/India/H225/2009) spanning 2,500 nucleotides (from 134 to 2,633) were cloned and sequenced. BLAST results showed that these genes had a greater than 97% nucleotide sequence identity with different human JEV isolates from India. Phylogenetic analysis based on E- and C/prM genes indicated that the equine JEV isolate belonged to genotype III and was closely related to the Vellore group of JEV isolates from India.

Comparison of four diagnostic methods for detecting rabies viruses circulating in Korea

It is essential to rapidly and precisely diagnose rabies. In this study, we evaluated four diagnostic methods, indirect fluorescent antibody test (FAT), virus isolation (VI), reverse transcriptase polymerase chain reaction (RT-PCR), and rapid immunodiagnostic assay (RIDA), to detect rabies in animal brain homogenates. Out of the 110 animal brain samples tested, 20 (18.2%) were positive for rabies according to the FAT. Compared to the FAT, the sensitivities of VI, RT-PCR, and RIDA were 100, 100, and 95%, respectively. The specificities of VI, RT-PCR and RIDA were found to be 100, 100, and 98.9%, respectively. Rabies viruses circulating in Korea were isolated and propagated in murine neuroblastoma (NG108-15) cells with titers ranging from 101.5 to 104.5 TCID50/mL. Although the RIDA findings did not completely coincide with results obtained from FAT, VI, and RT-PCR, RIDA appears to be a fast and reliable assay that can be used to analyze brain samples. In summary, the results from our study showed that VI, RT-PCR, and RIDA can be used as supplementary diagnostic tools for detecting rabies viruses in both laboratory and field settings.

Establishment and characterization of an infectious cDNA clone of a classical swine fever virus LOM strain

Classical swine fever virus (CSFV) causes a highly contagious disease among swine that has an important economic impact worldwide. CSFV strain LOM is an attenuated virus of low virulent strain of Miyagi isolated from Japan in 1956. Eight DNA fragments representing the genome of the CSFV strain LOM were obtained by RT-PCR. These were used to determine the complete nucleotide sequence and construct a full-length cDNA clone which was called Flc-LOM. Sequence analysis of the recombinant clone (Flc-LOM) revealed the presence of eight mutations, resulting in two amino acid substitutions, when compared to the parental sequence. RNA transcripts of both LOM and Flc-LOM were directly infectious in PK-15 cells. The rescued Flc-LOM virus grew more slowly than the parental virus, LOM, in the cells. Intramuscular immunization with Flc-LOM was safe and highly immunogenic in pigs; no clinical signs or virus transmission to sentinel animals were observed after 35 days. CSFV-specific neutralizing antibodies were detected 14 days post-infection. After challenge with the virulent CSFV strain SW03, pigs immunized with Flc-LOM were shown to be fully protected. Thus, our newly established infectious clone of CSFV, Flc-LOM, could serve as a vaccine candidate.

Detection of avian leukosis virus subgroup J in albumen of commercial and native fowl eggs using RT-PCR in Fars province of Iran

The subgroup J of ALV [ALV-J] has emerged as an important pathogen of meat-type chickens since 1989. This virus is responsible for economic losses due to both mortality and depressed performance in chickens. So, the objective of this study is the detection of ALV-J in the albumen of commercial and native fowl eggs using RT-PCR. Three hundred and seventy egg albumens were randomly selected from different farms of Fars province, Iran. These eggs were obtained from the flocks of two research centers on native fowl production [70 eggs], a broiler grandparent farm [60 eggs], three broiler breeder farms [180 eggs], and a commercial layer flock [60 eggs]. RT-PCR was undertaken on isolated RNA from egg samples using a pair of ALV-J specific primers H5/H7 that produced a 545 basepair fragment. RT-PCR analyses detected ALV-J in 15 of 180 [8.33%] samples from three broiler breeders farms, 17 of 70 [24.28%] samples from flocks of two research centers of native fowls production, and none of the samples of commercial layer and broiler grandparent farms. Direct sequencing using primers specific for subgroup ALV-J verified the viral subgroup in the RT-PCR amplification products. This is the first report of the ALV-J in egg albumen in Iran which indicates the necessity to apply eradication programs for ALV-J in the poultry industry and native fowls in Iran

The critical time of avian leukosis virus subgroup J-mediated immunosuppression during early stage infection in specific pathogen-free chickens

The critical time of avian leukosis virus subgroup J (ALV-J)-mediated immunosuppression was determined by body weight, relative immune organ weight, histopathology, and presence of group specific antigen and antibodies in specific pathogen-free (SPF) chickens. CD4+ and CD8+ cell activity in the spleen, total and differential leukocyte counts in blood, and viral RNA levels in spleen were measured. Significant growth suppression was observed in the two ALV-J-infected groups. A strong immune response by infected groups was present in spleen at 2-weeks-of-age, but after 4-weeks-of-age, the response decreased quickly. The thymus and bursa showed persistent immunosuppression until 4-weeks-of-age. Proliferation of fibroblasts and dendritic cells were observed in immune organs at 4- and 5-weeks-of-age. However, the granulocyte cell number was markedly lower in the infected groups than in the control group. In group 1 (day 1 infection) CD4+ cells increased during the second week but significantly decreased during the fourth week, while group 2 (day 7 infection) showed the opposite effect. Viral RNA increased significantly by the fourth week. These data identify 3~4 weeks post-infection as the key time at which the ALV-J virus exerts its immunosuppressive effects on the host.

Ocorrência de animais persistentemente infectados pelo vírus da diarréia viral bovina em rebanhos bovinos nos Estados de Minas Gerais e São Paulo / Occurrence of persistently infected animals with bovine viral diarrhoea virus in cattle herds from the States of Minas Gerais and São Paulo, Brazil

A pesquisa de animais persistentemente infectados (PI) pelo vírus da diarréia viral bovina (BVDV) foi realizada em 26 rebanhos bovinos, não vacinados contra o BVDV, localizados nos Estados de Minas Gerais e São Paulo, Brasil. Utilizando uma estratégia de amostragem, de cada rebanho foram obtidas cinco amostras de sangue de bezerros, entre 6 e 12 meses de idade, e os soros sanguíneos foram submetidos ao teste de virusneutralização (VN) para o BVDV-1 e o BVDV-2. Os rebanhos que apresentaram pelo menos três das cinco amostras reagentes a um dos genótipos do BVDV, e com títulos de anticorpos superiores a 128, foram selecionados para a pesquisa de animais PI. Em três rebanhos que apresentaram tal condição, foram colhidas amostras pareadas de sangue de todos os bovinos do rebanho, com intervalo de 30 dias entre as colheitas, e o soro sanguíneo foi submetido ao teste de VN para o BVDV-1 e o BVDV-2. Nas amostras não reagentes a pelo menos um dos genótipos do BVDV e naquelas provenientes de bovinos com menos de seis meses de idade, realizou-se a pesquisa do BVDV pela reação em cadeia da polimerase precedida pela transcrição reversa (RT-PCR). Dos rebanhos analisados, foram detectados dois animais PI a partir de amostras obtidas nas colheitas pareadas provenientes de um rebanho localizado no Estado de Minas Gerais.

The research on persistently infected (PI) animals with bovine viral diarrhoea virus (BVDV) was conducted in 26 cattle herds, which were not BVDV vaccinated, located in the states of Minas Gerais and São Paulo, Brazil. Using a sampling strategy, five samples of blood were collected from 6 to 12-month-old calves of each herd, and the blood sera were tested by virusneutralization test (VN) to BVDV-1 and BVDV-2. The herds that had at least three out of five samples reacting to one of the genotypes of BVDV and antibody titers greater than 128 were selected to PI animals research. In three of the herds that matched the before-mentioned criteria, paired blood samples were collected from all its individuals considering a collection interval of 30 days. The blood sera of these samples were VN tested against BVDV-1 and BVDV-2. In samples not reacting to at least one of the BVDV genotypes and also in those collected from calves of less than six months of age, virus research was undertaken by reverse transcription polymerase chain reaction (RT-PCR). From the examined herds, two PI animals were detected in paired samples obtained from a herd located in the state of Minas Gerais.

Serologic evidence of the recent circulation of Saint Louis encephalitis virus and high prevalence of equine encephalitis viruses in horses in the Nhecolândia sub-region in South Pantanal, Central-West Brazil

As in humans, sub-clinical infection by arboviruses in domestic animals is common; however, its detection only occurs during epizootics and the silent circulation of some arboviruses may remain undetected. The objective of the present paper was to assess the current circulation of arboviruses in the Nhecolândia sub-region of South Pantanal, Brazil. Sera from a total of 135 horses, of which 75 were immunized with bivalent vaccine composed of inactive Eastern equine encephalitis virus (EEEV) and Western equine encephalitis virus(WEEV) and 60 were unvaccinated, were submitted to thorough viral isolation, reverse transcriptase polymerase chain reaction (RT-PCR) and neutralization tests for Saint Louis encephalitis virus (SLEV), EEEV, WEEV and Mayaro virus (MAYV). No virus was isolated and viral nucleic-acid detection by RT-PCR was also negative. Nevertheless, the prevalence of neutralizing antibodies in horses older than seven months was 43.7 percent for SLEV in equines regardless of vaccine status, and 36.4 percent for WEEV and 47.7 percent for EEEV in unvaccinated horses. There was no evidence of MAYV infections. The serologic evidence of circulation of arboviruses responsible for equine and human encephalitis, without recent official reports of clinical infections in the area, suggests that the Nhecolândia sub-region in South Pantanal is an important area for detection of silent activity of arboviruses in Brazil.