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Sapovirus (genus Sapovirus, family Caliciviridae), along with norovirus, is recognized as an important cause of acute intestinal infection (AII) in humans. Human sapovirus strains exhibit significant genetic diversity. The existence of 18 genotypes, combined into four genogroups (GI, GII, GIV, GV), has been shown. Reverse transcription-PCR (RT-PCR) techniques are widely used for the detection of sapoviruses in clinical samples due to their high sensitivity and broad reactivity. To further characterize the identified sapoviruses, genotyping is carried out, which is the basis for constructing a classification system for human sapoviruses. The purpose of this review was to highlight modern methods for the detection and genotyping of sapoviruses based on the reverse transcription polymerase chain reaction and viral genome sequencing. [ABSTRACT FROM AUTHOR]

Novel antigenic variant strains of the infectious bursal disease virus (IBDV) classified into genogroup A2d have been found in the western part of Japan since 2017. Novel antigenic variant IBDVs now occur in higher frequencies in poultry houses and have been detected in the eastern part of Japan, indicating the spread of IBDVs despite the usual IBDV vaccination. We isolated a novel antigenic variant IBDV, designated as the B2977CE2C3 strain. The B2977CE2C3 strain had two genogroup A2d specific amino acids—lysine and isoleucine, at 221 and 252 aa—along with the other genogroup A2 common amino acids in the projection domains of the VP2 protein corresponding to the virus-neutralizing epitopes and viral pathogenicity. Experimental infection of the B2977CE2C3 strain did not produce any apparent clinical signs in the specific-pathogen-free chickens during the observation period (21 days), but atrophy of the bursa of Fabricius (BF) was apparent. The mean BF to the body weight ratio was 0.35 in negative control chickens at 21 days post-infection (pi) but 0.06 in the B2977CE2C3 infected group. An extremely high copy number of the IBDV genome (>108 copies/µL) was observed in the BF at 3 days pi, while a high copy number of the IBDV genome (>106 copies/µL) was observed in the thymus, spleen cecal tonsil, and bone marrow even though macroscopic lesions were not apparent in these organs. [ABSTRACT FROM AUTHOR]

Background: The genus Sapovirus in the family Caliciviridae comprises of a genetically diverse group of viruses that are responsible for causing acute gastroenteritis in both human and animals globally. As the number of sequences continues to grow and more recombinant sequences are identified, the classification criteria of genogroups and genotypes of sapovirus need to be further refined. In this study, we aimed to optimize the classification of sapoviruses. Results: Through evolutionary clustering and genetic distance analysis, we have updated the classification criteria for VP1 genogroup and genotypes. We adjusted the original mean values ± 3 standard deviations (SD) of genetic distances to mean values ± 2.5SD, resulting the corresponding cutoff values for the same genotype and genogroup set at <0.161 and <0.503, respectively. Additionally, we established classification criteria for RdRp types and groups, referred to as P-types and P-groups,, with mean values ± 2SD and cutoff values of <0.266 and <0.531 for the same type and group, respectively. This refinement has expanded the VP1 genogroups to thirty-four and identified twenty-four P-groups. For human sapoviruses, the new criteria have resulted in the addition of one genotype, GV.PNA1. Moreover, the new criteria defined three P-groups and 21 P-types for human sapoviruses. Spatial-temporal analysis revealed no specific distribution pattern for human sapoviruses. Conclusions: We established a dual typing system on classification based on VP1 and RdRp nucleotide sequences for sapoviruses. [ABSTRACT FROM AUTHOR]

Novel antigenic variant strains of the infectious bursal disease virus (IBDV) classified into genogroup A2d have been found in the western part of Japan since 2017. Novel antigenic variant IBDVs now occur in higher frequencies in poultry houses and have been detected in the eastern part of Japan, indicating the spread of IBDVs despite the usual IBDV vaccination. We isolated a novel antigenic variant IBDV, designated as the B2977CE2C3 strain. The B2977CE2C3 strain had two genogroup A2d specific amino acids—lysine and isoleucine, at 221 and 252 aa—along with the other genogroup A2 common amino acids in the projection domains of the VP2 protein corresponding to the virus-neutralizing epitopes and viral pathogenicity. Experimental infection of the B2977CE2C3 strain did not produce any apparent clinical signs in the specific-pathogen-free chickens during the observation period (21 days), but atrophy of the bursa of Fabricius (BF) was apparent. The mean BF to the body weight ratio was 0.35 in negative control chickens at 21 days post-infection (pi) but 0.06 in the B2977CE2C3 infected group. An extremely high copy number of the IBDV genome (>108 copies/µL) was observed in the BF at 3 days pi, while a high copy number of the IBDV genome (>106 copies/µL) was observed in the thymus, spleen cecal tonsil, and bone marrow even though macroscopic lesions were not apparent in these organs.

Infectious bursal disease (IBD) is an acute, highly contagious, and immunosuppressive disease of chickens resulting in global economic losses to the poultry industry despite extensive vaccination. The emergence of very virulent IBDVs in global epizootics, including those from Egypt, demonstrates how occasionally occurring genetic changes lead to simultaneous evolution of distinct IBDV strains across the world. Upon phylogenetic analysis of Egyptian IBDVs, virus strains from chickens and turkey (n= 146) were clustered within genogroup 3, whereas, twenty nine isolates were closely related to vaccinal strains in genogroup 1. Regarding to sequences from wild birds (cattle egret and green winged teal) were clustered within G1a (n=2) and G3a (n=1) subgen groups. The highest IBDV percentages during 2013-2022 was in 2020 (n=71; 20%) and in Behera Province (n= 54; 15.3%) followed by Sharkia Province (n= 51; 14.4%). The data revealed the characteristic molecular traits of IBDVs circulating in Egypt between 2013 and 2022, which will help in development of effective vaccines subsequently disease prevention. In addition to, it offers helpful insights for carrying out further surveys that are required to increase the understanding of IBDV prevalence in wild migratory and free-living birds. [ABSTRACT FROM AUTHOR]

ABSTRACT: Infectious bursal disease (IBD) is a viral disease that affects the ability of chickens to produce humoral immune responses. One way to prevent the disease is the passage of maternally derived antibodies (MDA) from dams to offsprings via the yolk. Despite sanitary measures, which include immunization with genogroup 1 (G1) vaccines, infections with IBDV genogroup 4 (G4) in young animals have been detected. The aim of this study was to determine whether a local IBDV isolate belonging to G4 could evade the immunity generated by MDAs. Twelve-day-old animals positive for MDA, were inoculated with G1 or G4 isolates or phosphate buffered saline (PBS) as a control. After 1 wk, the animals were sacrificed and the following parameters were evaluated: bursa-body (BB) ratio, viral load, and histologic damage in the bursa of Fabricius. Results showed that G4-infected animals had significant differences in the BB ratio compared to the PBS group. In addition, viral load was significantly higher in the G4 group than in the G1 group. Histologic damage in the bursa of Fabricius was detected only in G4-infected MDA chickens. Our results suggest that infection with G4 local isolate can circumvent the immunity generated by MDA and, furthermore, that G4 isolate does not differ in its pathogenicity from G1 isolate, which underlines the need to include variant strains in vaccine formulations to reduce potential losses caused by these viruses.

The role of viruses in the diarrhea algorithm is very important. In addition, losses due to diarrhea are quite high for calves, and herd control and protection cannot be performed in cases where a rapid etiological diagnosis cannot be made. Group A rotaviruses and coronaviruses, which are the main viral etiological agents of enteric diseases in calves, are frequently detected in these infections. However, the presence/prevalence of other agents in single or mixed infections is still up-to-date and under investigation. Two genetically distinct bovine enteric caliciviruses are known: genogroup III noroviruses (NoVsGIII), which are genetically related to human noroviruses, and neboviruses, which represent a new genus of caliciviruses. In this study, it was aimed to elucidate the role and genotypes of noroviruses (bovine norovirus) in the diarrheal paradigm in calves. In order to determine the presence of norovirus and genogroups, a total of 92 diarrheal stools from calves were included in the study from the collection material in our laboratory. In the study, reverse transcription nested polymerase chain reaction was performed with specific primer pairs. Polymerase chain reaction (PCR) test results were positive in 3 samples (3/92, 3.2%) and were subjected to sequence analysis. According to the results of bioinformatics analysis, noroviruses are divided into 7 main genogroups in light of recent literature. Among these genogroups, bovine noroviruses were included in genogroup III (GIII). There are also subtypes within GIII. Of the 3 strains identified in our study, 2 were grouped in GIII-2a (TR/ERZ/25 and TR/ELZ/23) and 1 strain was grouped in GIII-2b (TR/ERZ/9). Conducting more comprehensive molecular prevalence studies on noroviruses, focusing on the detected genogroups in vaccine studies to be developed and determining their prevalence, the effects on the pathogenesis and clinical picture should be revealed. Determination of genogroup type profiles in the world and in our country and, if vaccines can be developed, elucidating different genogroup types on protection will be beneficial in diarrhea and norovirus dilemmas. [ABSTRACT FROM AUTHOR]

Piscirickettsiosis, the cause of great losses in Chilean salmonid population, is produced by Piscirickettsia salmonis. The bacterium has developed resistance to treatments over time, including antibiotics and vaccines, due to a variety of evasive strategies, including biofilm formation. The bacterium has genomic islands, clusters of relevant genes that vary between genomes. This work aimed to analyze the relationship between number of pathogenic islands and their virulence genes in complete genomes from different strains of EM and LF genogroups, besides the search for genomic islands with non-virulent factors, like metabolic islands and fitness. Furthermore, we evaluated conservation and variation between strains from different genogroup, considering LF-89 (ATCC) and Psal-001 as reference strains, analyzing genomes through averages by two bioinformatic tools. About 13 pathogenic islands/strain were quantified, with 1 to 47 loci/island with unique behaviors for genogroup. Comparing islands regarding genomes, higher conservation rate was seen in EM genogroup, against oscillating values within LF genogroup strains. Non-virulent factors were also found. Overall, EM genogroup showed greater convergence in island conservation between strains concerning the pathogenic function compared to LF strains. Finally, different transposases were found to support high genomic plasticity and the scarcity of cured genes complicated correct identification.

CSF is enzootic in most of pig-producing states, particularly in the NorthEastern (NE) region of India. In this study, a total of 249 sera and 190 tissue samples were collected from different parts of Meghalaya. Samples were processed by ELISA and RT-PCR for serological and molecular diagnosis. Representative positive samples from the Khasi Hills region were selected for sequencing and “close grid” phylogenetic relationship using partial genomic regions of 5′UTR and E2. High seroprevalence (74.7%) of CSFV was recorded. Detection of the CSFV genome in serologically positive serum samples and tissue samples was 61.29% and 18.94%, respectively. BLAST and phylogenetic analyses indicate the clustering of all the field samples in subgroup 2.2, with high identity with EF014334 from China. Molecular structural modelling of the E2 partial sequence using representative sequences MG563797 from Meghalaya and EF014334 from China indicate potential changes in the protein motif and its conformation, which may explain the emergence of subgroup 2.2 CSFV replacing the predominant subgroup 1.1 viruses in NorthEast India. The epidemiological information presented in this study may be helpful for determination of disease incidence in this region, whereas the virus profile may be useful for framing disease control programs.

The article presents the results of a molecular genetic study of two isolates of the Pseudorabies virus that were isolated from pigs in Ukraine. Bioinformatic analysis of the gE gene fragment of Aujeszky's disease virus (Pseudorabies virus) isolates was carried out in order to determine the phylogenetic relationships and homology of nucleotide sequences. Fragments of the Aujeszky disease virus genome corresponding to the C-terminal region of the gE gene were selected for sequencing and further analysis. As a result of the conducted studies, it was demonstrated that the nucleotide sequences of the analyzed samples differ from each other by the presence of ACG insert in the tandem repeats region. Comparison of the studied sequences with the sequences of strains/isolates of the Aujeszky's disease virus found in Europe and Asia, presented in the GenBank database, indicates that such an insert is characteristic for the Min-A and HNJZ strains (position 1487 in the gE gene) isolated in Asia. Analysis of the homology of nucleotide sequences showed that the sequence of the gE gene fragment of sample No. 1 is 100% identical to the sequences of strains 89V87 and 00V72 isolated in Belgium. The homology of the nucleotide sequence of the gE gene fragment of sample No. 3 with strains 89V87 and 00V72 was 99.13%. In order to clarify the analyzed samples belonging to a particular genogroup (genetic cluster), a phylogenetic dendrogram was constructed. This demonstrates the phylogenetic relationships between strains/isolates of the Aujeszky's disease virus. It was found that the analyzed samples belong to the genetic cluster uniting European strains/isolates, and the studied isolates are most genetically close to strains 89V87 and 00V72.

The enteric chicken astrovirus (CAstV) and avian nephritis virus (ANV) are the type species of the genus Avastrovirus (AAstV; Astroviridae family), capable of causing considerable production losses in poultry. Using next-generation sequencing of a cloacal swab from a backyard chicken in Tanzania, we assembled genome sequences of ANV and CAstV (6918 nt and 7318 nt in length, respectively, excluding poly(A) tails, which have a typical AAstV genome architecture (5′-UTR-ORF1a-ORF1b-ORF2-'3-UTR). They are most similar to strains ck/ANV/BR/RS/6R/15 (82.72%) and ck/CAstV/PL/G059/14 (82.23%), respectively. Phylogenetic and sequence analyses of the genomes and the three open reading frames (ORFs) grouped the Tanzanian ANV and CAstV strains with Eurasian ANV-5 and CAstV-Aii viruses, respectively. Compared to other AAstVs, the Tanzanian strains have numerous amino acid variations (substitutions, insertions and deletions) in the spike region of the capsid protein. Furthermore, CAstV-A has a 4018 nt recombinant fragment in the ORF1a/1b genomic region, predicted to be from Eurasian CAstV-Bi and Bvi parental strains. These data should inform future epidemiological studies and options for AAstV diagnostics and vaccines. [ABSTRACT FROM AUTHOR]

Pathogenic strains of infectious bursal disease virus (IBDV) are associated with increased morbidity, mortality, and immunosuppression in susceptible chickens. Backyard poultry is increasing in popularity in the United States, but very little is known about the prevalence and molecular epidemiology of IBDV within these flocks. We performed a retrospective study and phylogenetic analyses of IBDV detected in backyard chickens (BYCs) submitted to the California Animal Health and Food Safety (CAHFS) diagnostic laboratory system in 2009-2017. There were 17 CAHFS autopsy cases of very virulent IBDV (vvIBDV) segment A detected by RT-rtPCR in BYC flocks from 7 counties in California from 2009-2017. During this same time period, non-vvIBDV genotypes were detected by RT-rtPCR in 16 autopsy cases originating from BYC premises in 10 counties in California. Subsequent RT-PCR and phylogenetic analysis of a segment of the hvVP2 and VP1 gene identified vvIBDV, interserotypic reassortant IBDV (vvIBDV segment A and serotype 2 segment B), and non-vvIBDV (variant/subclinical IBDV and classic IBDV) strains in BYC flocks in California.

The article presents the results of a molecular genetic study of two isolates of the Pseudorabies virusthat were isolated from pigs in Ukraine. Bioinformatic analysis of the gE gene fragment of Aujeszky's disease virus (Pseudorabies virus) isolates was carried out in order to determine the phylogenetic relationships and homology of nucleotide sequences. Fragments of the Aujeszky disease virus genome corresponding to the C-terminal region of the gE gene were selected for sequencing and further analysis. As a result of the conducted studies, it was demonstrated that the nucleotide sequences of the analyzed samples differ from each other by the presence of ACG insert in the tandem repeats region. Comparison of the studied sequences with the sequences of strains/isolates of the Aujeszky's disease virusfound in Europe and Asia, presented in the GenBank database, indicates that such an insert is characteristic for the Min-A and HNJZ strains (position 1487 in the gE gene) isolated in Asia. Analysis of the homology of nucleotide sequences showed that the sequence of the gE gene fragment of sample No. 1 is 100% identical to the sequences of strains 89V87 and 00V72 isolated in Belgium. The homology of the nucleotide sequence of the gE gene fragment of sample No. 3 with strains 89V87 and 00V72 was 99.13%. In order to clarify the analyzed samples belonging to a particular genogroup (genetic cluster), a phylogenetic dendrogram was constructed. This demonstrates the phylogenetic relationships between strains/isolates of the Aujeszky's disease virus. It was found that the analyzed samples belong to the genetic cluster uniting European strains/isolates, and the studied isolates are most genetically close to strains 89V87 and 00V72 [ABSTRACT FROM AUTHOR]

The emergence of Neisseria gonorrhoeae isolates displaying resistance to antimicrobials, in particular to ceftriaxone monotherapy or ceftriaxone plus azithromycin, represents a global public health concern. This study aimed to analyze the trend of antimicrobial resistance in a 7-year isolate collection retrospective analysis in Italy. Molecular typing on a subsample of gonococci was also included. A total of 1,810 culture-positive gonorrhea cases, collected from 2013 to 2019, were investigated by antimicrobial susceptibility, using gradient diffusion method, and by the N. gonorrhoeae multiantigen sequence typing (NG-MAST). The majority of infections occurred among men with urogenital infections and 57.9% of male patients were men who have sex with men. Overall, the cefixime resistance remained stable during the time. An increase of azithromycin resistance was observed until 2018 (26.5%) with a slight decrease in the last year. In 2019, gonococci showing azithromycin minimum inhibitory concentration above the EUCAST epidemiological cutoff value (ECOFF) accounted for 9.9%. Ciprofloxacin resistance and penicillinase-producing N. gonorrhoeae (PPNG) percentages increased reaching 79.1% and 18.7% in 2019, respectively. The most common sequence types identified were 5,441, 1,407, 6,360, and 5,624. The predominant genogroup (G) was the 1,407; moreover, a new genogroup G13070 was also detected. A variation in the antimicrobial resistance rates and high genetic variability were observed in this study. The main phenotypic and genotypic characteristics of N. gonorrhoeae isolates were described to monitor the spread of drug-resistant gonorrhea. [ABSTRACT FROM AUTHOR]

CSF is enzootic in most of pig-producing states, particularly in the NorthEastern (NE) region of India. In this study, a total of 249 sera and 190 tissue samples were collected from different parts of Meghalaya. Samples were processed by ELISA and RT-PCR for serological and molecular diagnosis. Representative positive samples from the Khasi Hills region were selected for sequencing and "close grid" phylogenetic relationship using partial genomic regions of 5′UTR and E2. High seroprevalence (74.7%) of CSFV was recorded. Detection of the CSFV genome in serologically positive serum samples and tissue samples was 61.29% and 18.94%, respectively. BLAST and phylogenetic analyses indicate the clustering of all the field samples in subgroup 2.2, with high identity with EF014334 from China. Molecular structural modelling of the E2 partial sequence using representative sequences MG563797 from Meghalaya and EF014334 from China indicate potential changes in the protein motif and its conformation, which may explain the emergence of subgroup 2.2 CSFV replacing the predominant subgroup 1.1 viruses in NorthEast India. The epidemiological information presented in this study may be helpful for determination of disease incidence in this region, whereas the virus profile may be useful for framing disease control programs. [ABSTRACT FROM AUTHOR]

Background and Aim: Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea in suckling piglets, leading to severe economic losses in the swine industry. Commercial vaccines have limited effectiveness against different genogroups of PEDV and the shedding of virus. The C-terminal of the S1 domain and the N-terminal of the S2 domain (S1-2) protein of the spike (S) protein have four neutralizing epitopes. However, research on the expression of the S1-2 segment of the S gene has been limited. In this study, we expressed a recombinant S1-2 protein of the S protein of the PEDV Thai isolate and characterized the immunological properties of the recombinant S1-2 protein. Materials and Methods: The S1-2 segment of the S gene of the PEDV Thai isolate (G2b) was amplified, cloned into the pBAD202/D-TOPO® vector (Invitrogen, Carlsbad, CA, USA), and expressed in Escherichia coli. The optimum concentration of arabinose and the optimum induction time for the expression of the recombinant S1-2 protein were determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The immunogenic reactivity of the recombinant S1-2 protein was determined using Western blot analysis with rabbit polyclonal antibodies against the SM98 strain of PEDV (G1a). Results: The recombinant S1-2 segment of the S gene of the PEDV Thai isolate protein was cloned and the recombinant S1-2 protein was successfully expressed. The optimum concentration of arabinose and the optimum induction time for the induction of the recombinant S1-2 protein were 0.2% and 8 h, respectively. The recombinant S1-2 protein reacted specifically with both rabbit anti-histidine polyclonal antibodies and rabbit anti-PEDV polyclonal antibodies. Conclusion: The recombinant S1-2 protein reacted with rabbit anti-PEDV polyclonal antibodies induced by the different PEDV genogroup. Therefore, the recombinant S1-2 protein may be a useful tool for the development of a diagnostic test for PEDV or for a vaccine against PEDV.

Feline calicivirus (FCV) is a highly infectious pathogen that causes upper respiratory tract disease (URTD), but the enteric FCVs raise concerns regarding their role of an enteric pathogen. In this study, between 2019 and 2020, 101 clinical samples from domestic cats with symptoms of URTD, with or without enteritis, were collected for FCV‐specific detection. The FCV‐positive rate reached to 42.4% (28/66) in cats with respiratory symptoms. The rates were 11.1% (3/27) and 12.5% (1/8) when faeces and serum samples were measured using reverse transcription polymerase chain reaction (RT‐PCR), respectively. Ten FCV strains were successfully isolated from respiratory and enteric sources in domestic cats from Guangxi. Phylogenetic analysis based on the genome sequences of 11 isolates (including GX01‐13 isolated in 2013) indicated that the newly characterized FCV strains had two recombinant events in comparison with other FCVs and were of respiratory and enteric origins. These strains displayed high genetic diversity, and they were divided into two genogroups (I and II). Of these, the GXNN02‐19 isolate was grouped with previously published Chinese isolates that were identified as genogroup II, which contained three specific amino acid residues (377K, 539V and 557S) in the VP1 protein. In addition, the three enteric viruses appeared genetically heterogeneous to each other. All isolates were found to be more sensitive when exposed to low pH conditions, but they were resistant to treatment with trypsin and bile salts. Furthermore, there were no significant differences between the respiratory and enteric FCVs. Our results showed that the genetically distinct FCV strains with genogroups I and II from respiratory and enteric origins were co‐circulating in this geographical area. Also, it was revealed that the potential recombinant events between the enteric and respiratory FCVs suggested an important role of enteric FCV during the evolution. [ABSTRACT FROM AUTHOR]

Simple Summary: Infectious hematopoietic necrosis virus (IHNV) can cause severe disease and mortality in salmon and trout. Two major groups of the virus are found in the North American Columbia River Basin and Washington Coast, designated U and M. The U and M groups pose different risks depending on the fish host species. Here, we report the development of a new diagnostic test that can both detect the presence of IHNV and rapidly discriminate between the U and M groups. The new assay proved sensitive and specific when applied to free ranging, returning adult Pacific salmon. Infectious hematopoietic necrosis virus (IHNV) is an acute pathogen of salmonids in North America, Europe, and Asia that is phylogenetically classified into five major virus genogroups (U, M, L, E, and J). The geographic range of the U and M genogroup isolates overlap in the North American Columbia River Basin and Washington Coast region, where these genogroups pose different risks depending on the species of Pacific salmon (Oncorhynchus spp.). For certain management decisions, there is a need to both test for IHNV presence and rapidly determine the genogroup. Herein, we report the development and validation of a U/M multiplex reverse transcription, real-time PCR (RT-rPCR) assay targeting the IHNV nucleocapsid (N) protein gene. The new U/M RT-rPCR is a rapid, sensitive, and repeatable assay capable of specifically discriminating between North American U and M genogroup IHNV isolates. However, one M genogroup isolate obtained from commercially cultured Idaho rainbow trout (O. mykiss) showed reduced sensitivity with the RT-rPCR test, suggesting caution may be warranted before applying RT-rPCR as the sole surveillance test in areas associated with the Idaho trout industry. The new U/M assay had high diagnostic sensitivity (DSe > 94%) and specificity (DSp > 97%) in free-ranging adult Pacific salmon, when assessed relative to cell culture, the widely accepted reference standard, as well as the previously validated universal N RT-rPCR test. The high diagnostic performance of the new U/M assay indicates the test is suitable for surveillance, diagnosis, and confirmation of IHNV in Pacific salmon from the Pacific Northwest regions where the U and M genogroups overlap. [ABSTRACT FROM AUTHOR]

Abstract Background Ferlaviruses are important pathogens in snakes often associated with respiratory and neurological disease. The detection of ferlaviral RNA by PCR is considered to be the most reliable method for the diagnosis of infection. The PCRs that have been used most commonly for this purpose have not been properly assessed to determine their sensitivity, specificity and ability to detect the known genetic diversity of this group of viruses. The aim of this study was to compare three published PCR protocols so that a single method could be recommended to laboratories that perform this testing. Results Comparisons were carried out using cell culture isolates and tissues from snakes infected with specific virus genotypes. A single round PCR targeting a short segment of the viral polymerase (L) gene provided the highest sensitivity and specificity, and detected isolated ferlaviruses from all four described genogroups, as well as from tissues of infected snakes. Conclusion A broadly-reactive PCR for the detection of all known ferlaviruses was found to provide a good combination of detection limit, specificity and speed. Based on these criteria, this method is recommended for the diagnosis of ferlavirus infections.

Feline noroviruses (FNoVs) are potential clinical pathogens in cats. To perform an epidemiological study of FNoV infection, it is necessary to develop a simple and effective method for virus detection. We investigated whether a commercial human NoV quantitative RT-PCR kit for the detection of human NoVs used in medical practice can be applied for FNoV detection. This kit was capable of detecting the FNoV gene regardless of the genogroup (GIV and GVI) in experimental and field samples. Based on the above findings, it is possible to detect FNoVs using human NoV tests. The relationship between FNoV infection and gastroenteritis in cats may be clarified by applying these methods to an epidemiological survey of FNoVs. [ABSTRACT FROM AUTHOR]

Feline calicivirus (FCV) is a highly infectious pathogen in cats and widely distributed worldwide with high genetic variation. Full-length open reading frame 2 of 5 from recently isolated Korean FCV isolates were sequenced and compared with those of global isolates. The results of phylogenetic analysis supported dividing global FCV isolates into two genogroups (type I and II) and demonstrated the presence of genogroup II in Korea, indicating their geographic spread in East Asia. High sequence variations in region E of the FCV isolates emphasizes that a novel vaccine needs to be developed to induce protective immunity against various FCV strains. [ABSTRACT FROM AUTHOR]

Sapoviruses (Caliciviridae) are considered important agents of gastroenteritis worldwide affecting animals and humans. In pig farming, the epidemiology is not completely understood because it can affect all stages of production, with symptomatic (diarrhea) or asymptomatic pigs. The aim of our study was to investigate Sapovirus occurrence in Brazilian pig farms. A total of 166 fecal samples of pigs, with different ages, from Minas Gerais, São Paulo, and Mato Grosso States were submitted to RT-PCR reactions and confirmed with nucleotide sequencing of Sapovirus RdRp gene. Six (3.61%) samples were positive and four had partial RdRp gene sequenced, putatively belonging to GVII.1 genogroup, also reported in swine herds in Brazil. [ABSTRACT FROM AUTHOR]

Norovirus (NoV) is one of the most important viral causes of acute gastroenteritis (AGE) in children worldwide. Only a few studies have reported AGE with NoV‐positive in some cities in Indonesia. This study aimed to investigate the incidence and clinical characteristic of NoV infection, and also genotype distribution of NoV in children with AGE in Jambi, as the capital and the largest city of Jambi province, Indonesia. Stool samples were collected from children (≤15 years of age) with AGE at three participating hospitals in Jambi from February to April 2019. The detection of NoV and its genotyping were carried out by reverse‐transcriptase polymerase chain reaction and direct sequencing. Of the 91 stool samples collected, 14 (15.4%) were positive for NoV. Fever, vomiting, and severe diarrhea were commonly observed in AGE with NoV, while level of dehydration was statistically significant difference between children with NoV‐positive and those with NoV‐negative. The most prevalent genotype was GI.4 (42.9%), followed by GII.6 (28.6%) and some other genotypes. Interestingly, this study found the predominance of GI.4, differed from previous reports in Indonesia. Continuously investigation of the circulating genotype is needed to control the NoV‐infected AGE. [ABSTRACT FROM AUTHOR]

Virus associated diarrhea remains one of the leading causes of children morbidity and mortality in Bangladesh. Human bocavirus (HBoV) has been reported as a potential pathogen of children's diarrhea worldwide. However, due to its frequent association with other gastroenteric pathogens, its role as diarrhea causative agent remains to be defined. This study focuses to detect the incidence of HBoV and adenovirus (AdV) and to determine the molecular and epidemiological characteristics of HBoV and AdV. Between January 2015 to January 2019, 290 fecal specimens were collected from diarrheal children in Bangladesh. All fecal specimens were tested for HBoV and AdV by conventional polymerase chain reaction and sequencing methods. HBoV was detected in 7.24% (21 of 290) of the stool samples, as a sole virus in 71.42% (15 of 21) of the positive samples. AdV was detected in 4.82% (14 of 290) of the samples. The most common clinical symptoms of HBoV infected patients were diarrhea (100%) and vomiting (57%). All of the isolates of HBoV were from HBoV1 and AdV were from AdV41, AdV5, AdV7, and AdV8. To the best of our knowledge, this is the first epidemiological and molecular analysis report of HBoV from clinical specimens in Bangladesh. In the future, more studies are needed to clarify the role of HBoV as diarrheal pathogens. Research Highlights: HBoV was detected in 7.24% (21 of 290) of the stool samples, as a sole virus in 71.42% (15 of 21) of the positive samples.HBoV was detected in 7.24% (21 of 290) of the stool samples, as a sole virus in 71.42% (15 of 21) of the positive samples.This is the first epidemiological and molecular analysis report of HBoV from clinical specimens in Bangladesh. [ABSTRACT FROM AUTHOR]

Introduction: Infectious pancreatic necrosis virus belongs to the genus Aquabirnavirus and family Birnaviridae. By VP2 gene similarity, aquatic birnavirus is clustered into seven genogroups. The aim of this study was to genetically analyse IPN viruses occurring on Polish fish farms. Materials and Methods: Samples from freshwater fish mostly from 2012 to 2013 and from northern Poland were examined for the presence of IPN virus using isolation on cell cultures, real‐time RT‐PCR and RT‐PCR. Fragments of 1,377 and 1,079 bp of the VP2 and VP5 genes, respectively, were sequenced, and the results were assembled into one consensus and analysed by Geneious software. The same VP2 gene region was compared and a phylogenetic tree generated by the neighbour‐joining method and MEGA6 software. Results: All tested Polish isolates belonged to genogroup 5, like other European Spajurup isolates. Conclusion: Our findings prove that there is only one IPN virus genogroup in Poland. Polish isolates show close relationships with each other. There is a close relationship between Polish isolates and isolates from Turkey, Spain and Iran. Isolate 57 is a separate branch related to isolates from the United States and Taiwan. This points to the likelihood of past virus introduction via import of stock from those countries. [ABSTRACT FROM AUTHOR]

Porcine epidemic diarrhoea virus (PEDV) belongs to the family Coronavirus, a genus of coronavirus, a highly contact‐infectious intestinal disease pathogen. In this study, we downloaded 62 PEDV S gene sequences uploaded to GenBank, including 10 uploaded by our laboratory from 2018, and constructed a PEDV S gene evolution tree using MEGA V7.0 software. Phylogenetic tree analysis indicated that the genogroup of PEDV in Sichuan Province was divided into three coexisting genogroups (GII‐a, GII‐b and GI‐a), of them, GII‐a has become the main genogroup in the province due to its prevalence and range of spread. Amino acid sequence analysis showed that there were amino acid insertions and deletions in the S protein encoded by the amplified S gene, and there were amino acid mutations in the COE and SS6 of the epitope in the amplified S protein. These results provide a basic research theory for understanding the prevalence of PEDV variation and controlling PED in Sichuan. [ABSTRACT FROM AUTHOR]

The Stenotrophomonas maltophilia complex (Smc) comprises opportunistic environmental Gram-negative bacilli responsible for a variety of infections in both humans and animals. Beyond its large genetic diversity, its genetic organization in genogroups was recently confirmed through the whole-genome sequencing of human and environmental strains. As they are poorly represented in these analyses, we sequenced the whole genomes of 93 animal strains to determine their genetic background and characteristics. Combining these data with 81 newly sequenced human strains and the genomes available from RefSeq, we performed a genomic analysis that included 375 nonduplicated genomes with various origins (animal, 104; human, 226; environment, 30; unknown, 15). Phylogenetic analysis and clustering based on genome-wide average nucleotide identity confirmed and specified the genetic organization of Smc in at least 20 genogroups. Two new genogroups were identified, and two previously described groups were further divided into two subgroups each. Comparing the strains isolated from different host types and their genogroup affiliation, we observed a clear disequilibrium in certain groups. Surprisingly, some antimicrobial resistance genes, integrons, and/or clusters of attC sites lacking integron-integrase (CALIN) sequences targeting antimicrobial compounds extensively used in animals were mainly identified in animal strains. We also identified genes commonly found in animal strains coding for efflux systems. The result of a large whole-genome analysis performed by us supports the hypothesis of the putative contribution of animals as a reservoir of Stenotrophomonas maltophilia complex strains and/or resistance genes for strains in humans. IMPORTANCE Given its naturally large antimicrobial resistance profile, the Stenotrophomonas maltophilia complex (Smc) is a set of emerging pathogens of immunosuppressed and cystic fibrosis patients. As it is group of environmental microorganisms, this adaptation to humans is an opportunity to understand the genetic and metabolic selective mechanisms involved in this process. The previously reported genomic organization was incomplete, as data from animal strains were underrepresented. We added the missing piece of the puzzle with whole-genome sequencing of 93 strains of animal origin. Beyond describing the phylogenetic organization, we confirmed the genetic diversity of the Smc, which could not be estimated through routine phenotype- or matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF)-based laboratory tests. Animals strains seem to play a key role in the diversity of Smc and could act as a reservoir for mobile resistance genes. Some genogroups seem to be associated with particular hosts; the genetic support of this association and the role of the determinants/corresponding genes need to be explored. [ABSTRACT FROM AUTHOR]

Bursa tissue samples from a pullet flock in New York State that was experiencing immune suppression related disease were sent to our laboratory in 2018. A very virulent infectious bursal disease virus (vvIBDV) was identified in those samples through molecular and pathogenicity studies and designated 1/chicken/USA/1054NY/18. Phylogenetic analyses of the hypervariable VP2 nucleotide sequence region indicated that this strain belonged to genogroup 3 which comprises the vvIBDV. Partial sequence data of the VP1 gene indicated this virus also had a VP1 typical of vvIBDV. While vvIBDV have previously been identified in the United States in California and Washington State, the 1054NY vvIBDV was most closely related to isolates from Ethiopia, suggesting it is a new introduction into the U.S. The 1054NY vvIBDV was used to challenge four-week old specific-pathogen-free (SPF) layer chicks where it caused 100% morbidity and 68.7% mortality within 4 days. Upon necropsy, gross pathological findings in infected SPF birds included small yellowish coloured bursas, some with haemorrhages on the serosal and mucosal surfaces. Microscopic lesions included inflammation, severe lymphocyte necrosis, atrophy of the follicles and follicular depletion of lymphocytes. RESEARCH HIGHLIGHTS A very virulent infectious bursal disease virus (vvIBDV) was detected in a pullet flock in New York state, USA. Nucleotide sequence analysis of the vvIBDV VP2 gene indicates it is not related to previous US vvIBDV isolates and appears to be a new introduction into the US. The New York vvIBDV caused 100% morbidity and 68.7% mortality in four-week-old specific-pathogen-free chicks. [ABSTRACT FROM AUTHOR]

• Five distinct tandem repeat of E. canis were found, indicating genetic diversity. • Two novel repetitions of the E. canis gp36 sequences were identified. • Our E. canis isolates fell into four well-defined phylogenetic clusters. Canine monocytic ehrlichiosis, an important tick-borne disease caused by Ehrlichia canis , is cosmopolitan but particularly prevalent in tropical and subtropical regions. In Turkey, the genetic diversity of E. canis remains undefined. The aim of this study was to characterize E. canis in naturally infected dogs from Turkey by sequencing and phylogenetic analysis of the Tandem Repeat Protein 36 (TRP36) encoded by the tr p36 gene. A total of 167 archived blood samples randomly collected from municipal shelter dogs in three distinct geographic regions were analyzed for E. canis. Only ten samples (5.98%) were found positive by PCR assays target regions of the tr p36 and 16S rRNA genes. Sequence analysis of Turkish E. canis TRP36 revealed five Tanden Repeat sequences (TRs) resulting to three TR genotypes: i) the previously reported US genotype composed exclusively from TRs of "TEDSVSAPA" sequence (14 or 8 TRs), ii) the previously Brazilian genotype composed exclusively from TRs of ASVVPEAE sequence (13 TRs), and iii) a novel genotype. In addition, phylogenetic analysis based on the entire sequences of TRP36 revealed that these genotypes correspond to four distinct genogroups (US genogroups I and II, Brazilian genogroup and Costa Rica-Turkey genogroup), all containing Turkish genotypes amongst other geographically distant E. canis genotypes. [ABSTRACT FROM AUTHOR]

Enterovirus 71 (EV-A71) is a major public health problem, causing a range of illnesses from hand-foot-and-mouth disease to severe neurological manifestations. EV-A71 strains have been phylogenetically classified into eight genogroups (A to H), based on their capsid-coding genomic region. Genogroups B and C have caused large outbreaks worldwide and represent the two canonical circulating EV-A71 subtypes. Little is known about the antigenic diversity of new genogroups as compared to the canonical ones. Here, we compared the antigenic features of EV-A71 strains that belong to the canonical B and C genogroups and to genogroups E and F, which circulate in Africa. Analysis of the peptide sequences of EV-A71 strains belonging to different genogroups revealed a high level of conservation of the capsid residues involved in known linear and conformational neutralization antigenic sites. Using a published crystal structure of the EV-A71 capsid as a model, we found that most of the residues that are seemingly specific to some genogroups were mapped outside known antigenic sites or external loops. These observations suggest a cross-neutralization activity of anti-genogroup B or C antibodies against strains of genogroups E and F. Neutralization assays were performed with diverse rabbit and mouse anti-EV-A71 sera, anti-EV-A71 human standards and a monoclonal neutralizing antibody. All the batches of antibodies that were tested successfully neutralized all available isolates, indicating an overall broad cross-neutralization between the canonical genogroups B and C and genogroups E and F. A panel constituted of more than 80 individual human serum samples from Cambodia with neutralizing antibodies against EV-A71 subgenogroup C4 showed quite similar cross-neutralization activities between isolates of genogroups C4, E and F. Our results thus indicate that the genetic drift underlying the separation of EV-A71 strains into genogroups A, B, C, E and F does not correlate with the emergence of antigenically distinct variants.

Gonorrhoea is one of the most common sexually transmitted infections and in 2012, the World Health Organization estimated about 78 million of new global urogenital cases among adults per year. The main concern during the latest decade has been the emergence and spread of multidrug-resistant strains of Neisseria gonorrhoeae. Resistance has emerged internationally to the extended-spectrum cephalosporins, ceftriaxone and cefixime, which are the last remaining options for empiric first-line monotherapy of gonorrhoea. In Poland, the levels of resistance to ciprofloxacin, benzylpenicillin and tetracycline are high, and the prevalence of azithromycin resistance has increased. However, no resistance to ceftriaxone has been identified. The currently spread multidrug-resistant strains frequently represent epidemic clones. The present paper reviews and describes the antimicrobial resistance and N. gonorrhoeae multiantigen sequence typing (NG-MAST) sequence types of N. gonorrhoeae strains spreading in Poland compared to the world. [ABSTRACT FROM AUTHOR]

Summary: In association with hepatitis B virus (HBV), hepatitis delta virus (HDV) is a subviral agent that may promote severe acute and chronic forms of liver disease. Based on the percentage of nucleotide identity of the genome, HDV was initially classified into three genotypes. However, since 2006, the original classification has been further expanded into eight clades/genotypes. The intergenotype divergence may be as high as 35%‐40% over the entire RNA genome, whereas sequence heterogeneity among the isolates of a given genotype is <20%; furthermore, HDV recombinants have been clearly demonstrated. The genetic diversity of HDV is related to the geographic origin of the isolates. This study shows the first comprehensive bioinformatic analysis of the complete available set of HDV sequences, using both nucleotide and protein phylogenies (based on an evolutionary model selection, gamma distribution estimation, tree inference and phylogenetic distance estimation), protein composition analysis and comparison (based on the presence of invariant residues, molecular signatures, amino acid frequencies and mono‐ and di‐amino acid compositional distances), as well as amino acid changes in sequence evolution. Taking into account the congruent and consistent results of both nucleotide and amino acid analyses of GenBank available sequences (recorded as of January, 2017), we propose that the eight hepatitis D virus genotypes may be grouped into three large genogroups fully supported by their shared characteristics. [ABSTRACT FROM AUTHOR]

This study evaluated different tissues of naturally contaminated oysters ( Crassostrea belcheri) for the presence of noroviruses. RNA from digestive tissues, gills, and mantle of the oysters was extracted and tested for norovirus genogroup (G) I, GII, and GIV using RT-nested PCR. In spiking experiments with a known norovirus, GII.4, the detection limits were 2.97 × 10 RNA copies/g of digestive tissues, 2.62 × 10 RNA copies/g of gills, and 1.61 × 10 RNA copies/g of mantle. A total of 85 oyster samples were collected from a fresh market in Bangkok, Thailand. Noroviruses were found in the oyster samples (40/85, 47%): GI (29/85, 34.1%), GII (9/85, 10.5%), mixed GI and GII (1/85, 1.2%), and GIV (1/85, 1.2%). All three genogroups were found in the digestive tissues of oysters. Norovirus GI was present in all three tissues with the highest frequency in the mantle, and was additionally detected in multiple tissues in some oysters. GII was also detected in all three tissues, but was not detected in multiple tissues in the same oyster. For genogroup I, only GI.2 could be identified and it was found in all tissues. For genogroup II, three different genotypes were identified, namely GII.4 which was detected in the gills and the mantle, GII.17 which was detected in the digestive tissues, and GII.21 which was detected in the mantle. GIV.1 was identified in the digestive tissues of one oyster. This is the first report on the presence of human GIV.1 in oyster in Thailand, and the results indicate oyster as a possible vehicle for transmission of all norovirus genogroups in Thailand. [ABSTRACT FROM AUTHOR]

Sapoviruses (SaV) are enteric viruses infecting humans and animals. SaVs are highly diverse and are divided into multiple genogroups based on structural protein (VP1) sequences. SaVs detected from pigs belong to eight genogroups (GIII, GV, GVI, GVII, GVIII, GIX, GX, and GXI), but little is known about the SaV genogroup distribution in the Japanese pig population. In the present study, 26 nearly complete genome (> 6000 nucleotide: nt) and three partial sequences (2429 nt, 4364 nt, and 4419 nt in length, including the entire VP1 coding region) of SaV were obtained from one diarrheic and 15 non-diarrheic porcine feces in Japan via a metagenomics approach. Phylogenetic analysis of the complete VP1 amino acid sequence (aa) revealed that 29 porcine SaVs were classified into seven genogroups; GIII (11 strains), GV (1 strain), GVI (3 strains), GVII (6 strains), GVIII (1 strain), GX (3 strains), and GXI (4 strains). This manuscript presents the first nearly complete genome sequences of GX and GXI, and demonstrates novel intergenogroup recombination events. [ABSTRACT FROM AUTHOR]

Background: Stenotrophomonas maltophilia is an opportunistic multi-drug-resistant bacterium responsible for healthcare-associated infections. Strategies for in-hospital infection control and management of carriers and environmental reservoirs remain controversial.Aim: To determine the population structure of S. maltophilia strains in hospitalized infected patients and to identify putative highly pathogenic subpopulations that require upgraded infection control measures.Methods: Eighty-three diverse human strains of various clinical origins from 18 geographically distant hospitals were characterized phenotypically and genotypically using a multi-locus sequence typing (MLST) approach.Findings: Neither a predominant nor emerging sequence type (ST) was identified. Among the 80 typeable strains, only 29% corresponded to described STs, especially ST5 (N=6) and ST4/26/31 (N=2). The ST distribution and the phylogenic tree based on the concatenated MLST genes did not account for geographical, clinical origin or antimicrobial susceptibility clustering. A phylogenic tree that included 173 ST profiles from the MLST database and the 80 typeable strains confirmed the high genetic diversity of S. maltophilia, the previously reported genogroup organization and the predominance of genogroup 6, as it represented 41% (33/80) of the strains. Unexpectedly, genogroup 2 was the second most prevalent genogroup and included 16% (13/80) of the strains. These genogroups represented 57% (20/35) of the strains in respiratory patients and 75% (9/12) of the strains in patients with cystic fibrosis.Conclusion: Beyond MLST, the over-representation of some genogroups among strains responsible for healthcare-associated infections was confirmed. Genogrouping affiliation is recommended to implement infection control measures selectively for the most pathogenic strains isolated from patient or environmental reservoirs. [ABSTRACT FROM AUTHOR]

We isolated an arbovirus from bovine blood in Indonesia. The arbovirus was obtained from the plasma of a cow showing no clinical symptoms in West Java in February 2014, and was identified as Akabane virus (AKAV) by AKAV-specific RT-PCR and subsequent sequence analysis. Phylogenetic analysis based on partial S segment indicated the AKAV isolate, WJ-1SA/P/2014, was most closely related with two isolates from Israel and Turkey reported in 2001 and 2015, respectively, and that WJ-1SA/P/2014 isolate belongs to AKAV genogroup Ib. This is the first isolation of AKAV from Indonesia. [ABSTRACT FROM AUTHOR]

The aim of this study was to determine the genetic diversity of Chinese feline calicivirus (FCV) isolates and their phylogenetic relationship with isolates from elsewhere in the world. Phylogenetic analysis was performed based on the partial open reading frame (ORF) 2 sequences (regions B–F) of 21 Chinese FCV isolates and 30 global isolates. The Chinese isolates included 13 isolates from Wuhan, which were isolated in this study, and eight previously published isolates. Sixteen Chinese isolates and two Japanese isolates formed a distinct phylogenetic cluster. Phylogenetic analysis based on the sequences of the complete genome, ORF1, ORF2 and ORF3 of selected isolates supported the above findings. Genogroup analysis revealed that FCV genogroup II is present in China. These findings suggest that Chinese FCV isolates are closely related to Japanese FCV isolates. [ABSTRACT FROM AUTHOR]

Porcine sapoviruses, members of the family Caliciviridae, have been considered as an aetiological agent of gastroenteritis in pigs. In this study, we analysed 251 faecal samples obtained from 3 to 90 day-old diarrhoeic pigs in the Czech Republic between January 2005 and June 2010 and tested them by negative staining electron microscopy for the presence of sapoviruses. Only one sample showed the presence of viral particles with characteristic sapovirus morphology. The presence of sapovirus (SaV) was confirmed by an RT-PCR assay with primers specific for the sapoviral RNA polymerase and capsid genes. Phylogenetic analysis based on a partial sequence of the RNA polymerase gene placed the new Czech isolate into the GVII genogroup of porcine sapoviruses; however, analysis of a portion of the capsid gene sequence classified the isolate as GIII of the genus Sapovirus. These contradictory findings indicate that recombinant porcine sapovirus was identified. According to our knowledge this is the first description of porcine sapovirus in domestic pigs in the Czech Republic

Ehrlichia canis and Babesia vogeli are vector-borne pathogens that infect blood cells and produce the diseases Canine Monocytic Ehrlichiosis (CME) and Babesiosis in dogs. Considering the lack of studies on these pathogens in Colombia, this study aims to determine the molecular prevalence and genetic characterization of E. canis and Babesia spp., in dogs from the Metropolitan Area of Bucaramanga (MAB), Santander, a region with one of the greatest pet densities in Colombia. One hundred eighty-five dogs were surveyed and analyzed through molecular, clinical, and hematological approaches. The molecular detection of E. canis and Babesia spp., was performed by conventional PCR targeting the dsb and 18S rRNA genes, respectively. To identify genogroups, E. canis positive samples underwent a hemi-nested PCR of the trp36 gene, and the PCR products were subsequently sequenced. Molecular analyses showed a prevalence of 13% (24/185; CI 95%, 8.1 – 18.0%) and 1.09% (2/185; CI 95,% -0.43 – 2.6%) for E. canis and B. vogeli respectively, as well as the presence of the genogroups US (USA), BR (Brazil), and CR (Costa Rica), in 62.5, 16.6, and 16.6% of E. canis positive samples, respectively. Values of hematocrit, hemoglobin, platelets, erythrocytes, white blood cell (WBC) count, lymphocytes, and eosinophils showed significant differences between animals infected with the different genogroups of E. canis (p < 0.05). In contrast, hematocrit values, hemoglobin, platelets, red blood cells, and creatine kinase MB isoenzyme (CK-MB) were lower in B. vogeli positive animals. Statistical analysis indicated that E. canis infection was associated with specific socioeconomic sectors as well as with some household features (p < 0.05). In conclusion, our results present evidence of the circulation of multiple genogroups of E. canis in the MAB, which is associated with different geographical origins and clinical traits. Epidemiological analyses suggest a need to increase molecular surveillance and prevention campaigns especially in lower socioeconomic sectors. [ABSTRACT FROM AUTHOR]

Sapovirus (SaV) is a causative agent of gastroenteritis. On the basis of capsid protein (VP1) nucleotide sequences, SaV can be divided into 5 genogroups (GI–GV), of which the GI, GII, GIV, and GV strains infect humans. SaV is uncultivable, but expression of recombinant VP1 in insect cells results in formation of viruslike particles (VLPs) that are antigenically similar to native SaV. In this study, we newly expressed SaV GII and GIV VLPs to compare genetic and antigenic relationships among all human SaV genogroups. Hyperimmune antiserum samples against VLPs reacted strongly with homologous VLPs. However, several antiserum samples weakly cross-reacted against heterologous VLPs in an antibody ELISA. Conversely, an antigen ELISA showed that VLPs of SaV in all human genogroups were antigenically distinct. These findings indicate a likely correspondence between SaV antigenicity and VP1 genogrouping and genotyping.

Immediate prevention of meningococcal disease relies in part on the prompt treatment with antibiotics of household and other close contacts of cases; however intervention with effective vaccination relies on identification of serogroup-causing strains. Parenteral antibiotic for patient with suspected meningococcal disease before hospital admission is currently recommended. Laboratory standard methods are hindered by failure to detect bacteria by this medical approach to improve patient prognosis. We assessed two polymerase chain reaction (PCR) assays to detect (crgA) and define the serogroups (siaD, orf-2, and ctrA) of Neisseria meningitidis in 120 cerebrospinal fluid (CSF) samples from positive cases (culture or antigen detection or direct smear). The PCR sensitivity for the identification of N. meningitidis was 100% (95% confidence interval, CI, 96-100%) compared to a sensitivity of 46% for culture (95% CI 37-55%), 61% for latex agglutination test (95% CI 52-70%), and 68% for Gram stain (95% CI 59-76%); PCR specificity was 97% (95% CI 82-100%). PCR correctly identified the serogroups A, B, C, W135, Y, and X in CSF samples with a sensitivity of 88% (95% CI 80-93%); the primer sets were 100% specific. The introduction of PCR-based assays shall increase laboratory confirmed cases, consequently enhancing surveillance of meningococcal disease.

Background Human Norovirus (NoV) is recognized as a major etiological agent of sporadic acute gastroenteritis worldwide. Objectives This study describes the clinical features associated with Human NoV occurrence in children and determines the prevalence and estimated viral burden of NoV in symptomatic and asymptomatic children in rural South Africa. Study design Between July 2014 and April 2015, outpatient children under 5 years of age from rural communities of Vhembe district, South Africa, were enrolled for the study. A total of 303 stool specimens were collected from those with diarrhea (n = 253) and without (n = 50) diarrhea. NoVs were identified using real-time one-step RT-PCR. Results One hundred and four (41.1%) NoVs were detected (62[59.6%] GII, 16[15.4%] GI, and 26[25%] mixed GI/GII) in cases and 18 (36%) including 9(50%) GII, 2(11.1%) GI and 7(38.9%) mixed GI/GII in controls. NoV detection rates in symptomatic and asymptomatic children (OR = 1.24; 95% CI 0.66–2.33) were not significantly different. Comparison of the median C T values for NoV in symptomatic and asymptomatic children revealed significant statistical difference of estimated GII viral load from both groups, with a much higher viral burden in symptomatic children. Conclusions Though not proven predictive of diarrhea disease in this study, the high detection rate of NoV reflects the substantial exposure of children from rural communities to enteric pathogens possibly due to poor sanitation and hygiene practices. The results suggest that the difference between asymptomatic and symptomatic children with NoV may be at the level of the viral load of NoV genogroups involved. [ABSTRACT FROM AUTHOR]

Several novel porcine bocaviruses (PBoVs) have been identified in pigs in recent years and association of these viruses with respiratory signs or diarrhea has been suggested. In this study, an EvaGreen ® -based multiplex real-time PCR (EG-mPCR) with melting curve analysis was developed for simultaneous detection and grouping of novel PBoVs into the same genogroups G1, G2 and G3. Each target produced a specific amplicon with a melting peak of 81.3 ± 0.34 °C for PBoV G1, 78.2 ± 0.37 °C for PBoV G2, and 85.0 ± 0.29 °C for PBoV G3. Non-specific reactions were not observed when other pig viruses were used to assess the EG-mPCR assay. The sensitivity of the EG-mPCR assay using purified plasmid constructs containing the specific viral target fragments was 100 copies for PBoV G1, 50 for PBoV G2 and 100 for PBoV G3. The assay is able to detect and distinguish three PBoV groups with intra-assay and inter-assay variations ranging from 0.13 to 1.59%. The newly established EG-mPCR assay was validated with 227 field samples from pigs. PBoV G1, G2 and G3 was detected in 15.0%, 25.1% and 41.9% of the investigated samples and coinfections of two or three PBoV groups were also detected in 25.1% of the cases, indicating that all PBoV groups are prevalent in Chinese pigs. The agreement of the EG-mPCR assay with an EvaGreen-based singleplex real-time PCR (EG-sPCR) assay was 99.1%. This EG-mPCR will serve as a rapid, sensitive, reliable and cost effective alternative for routine surveillance testing of multiple PBoVs in pigs and will enhance our understanding of the epidemiological features and possible also pathogenetic changes associated with these viruses in pigs. [ABSTRACT FROM AUTHOR]

To identify the prevalence and genetic variability of noroviruses in dogs, we tested fecal samples by using reverse transcription–PCR. We found canine norovirus in 40% and 9% of dogs with and without diarrhea, respectively. The virus was genetically unrelated to other noroviruses and constitutes a tentative new genogroup.

Aim: This study was undertaken with the aim to compare and establish the genetic relatedness between classical swine fever virus (CSFV) genogroup 2.2 isolate and pestivirus reference strains. Materials and Methods: The available complete genome sequences of CSFV/IND/UK/LAL-290 strain and other pestivirus reference strains were retrieved from GenBank. The complete genome sequence, complete open reading frame, 5' and 3' non-coding region (NCR) sequences were analyzed and compared with reference pestiviruses strains. Clustal W model in MegAlign program of Lasergene 6.0 software was used for analysis of genetic heterogeneity. Phylogenetic analysis was carried out using MEGA 6.06 software package. Results: The complete genome sequence alignment of CSFV/IND/UK/LAL-290 isolate and reference pestivirus strains showed 58.9-72% identities at the nucleotide level and 50.3-76.9% at amino acid level. Sequence homology of 5' and 3' NCRs was found to be 64.1-82.3% and 22.9-71.4%, respectively. In phylogenetic analysis, overall tree topology was found similar irrespective of sequences used in this study; however, whole genome phylogeny of pestivirus formed two main clusters, which further distinguished into the monophyletic clade of each pestivirus species. CSFV/IND/UK/LAL-290 isolate placed with the CSFV Eystrup strain in the same clade with close proximity to border disease virus and Aydin strains. Conclusion: CSFV/IND/UK/LAL-290 exhibited the analogous genomic organization to those of all reference pestivirus strains. Based on sequence identity and phylogenetic analysis, the isolate showed close homology to Aydin/04-TR virus and distantly related to Bungowannah virus. [ABSTRACT FROM AUTHOR]

Norovirus (NoV) has been classified into 6 genogroups, GI-GVI. In the present study, we identified novel feline NoV (FNoV) M49-1 strain. The C-terminal of RNA-dependent RNA polymerase of the FNoV M49-1 strain was highly homologous with GIV FNoV and GIV lion norovirus, whereas VP1 was highly homologous with GVI canine NoV (CNoV). Based on the results of the Simplot analysis, the FNoV M49-1 strain may have been produced by recombination between GIV.2 FNoV and GVI.1 CNoV. In addition, specific pathogen-free cats inoculated with FNoV gene-positive-fecal samples developed diarrhea symptoms, and the viral gene was detected in their feces and blood. [ABSTRACT FROM AUTHOR]