Genome analysis of Streptococcus spp. isolates from animals in pre-antibiotic era with respect to antibiotic susceptibility and virulence gene profiles.

Lyophilized Streptococcus spp. isolates (n = 50) from animal samples submitted to the diagnostic laboratory at the University of Connecticut in the 1940s were revivified to investigate the genetic characteristics using whole-genome sequencing (WGS). The Streptococcus spp. isolates were identified as follows; S. agalactiae (n = 14), S. dysgalactiae subsp. dysgalactiae (n = 10), S. dysgalactiae subsp. equisimils (n = 5), S. uberis (n = 8), S. pyogenes (n = 7), S. equi subsp. zooepidemicus (n = 4), S. oralis (n = 1), and S. pseudoporcinus (n = 1). We identified sequence types (ST) of S. agalactiae, S. dysgalactiae, S. uberis, S. pyogenes, and S. equi subsp. zooepidemicus and reported ten novel sequence types of those species. WGS analysis revealed that none of Streptococcus spp. carried antibiotic resistance genes. However, tetracycline resistance was observed in four out of 15 S. dysgalactiae isolates and in one out of four S. equi subsp. zooepidemicus isolate. This data highlights that antimicrobial resistance is pre-existed in nature before the use of antibiotics. The draft genome sequences of isolates from this study and 426 complete genome sequences of Streptococcus spp. downloaded from BV-BRC and NCBI GenBank database were analyzed for virulence gene profiles and phylogenetic relationships. Different Streptococcus species demonstrated distinct virulence gene profiles, with no time-related variations observed. Phylogenetic analysis revealed high genetic diversity of Streptococcus spp. isolates from the 1940s, and no clear spatio-temporal clustering patterns were observed among Streptococcus spp. analyzed in this study. This study provides an invaluable resource for studying the evolutionary aspects of antibiotic resistance acquisition and virulence in Streptococcus spp.

Simultaneous Deletion of the 9GL and UK Genes from the African Swine Fever Virus Georgia 2007 Isolate Offers Increased Safety and Protection against Homologous Challenge.

African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal viral disease of domestic pigs that has significant economic consequences for the swine industry. The control of African swine fever (ASF) has been hampered by the unavailability of vaccines. Successful experimental vaccines have been derived from naturally occurring, cell culture-adapted, or genetically modified live attenuated ASFV. Recombinant viruses harboring engineered deletions of specific virulence-associated genes induce solid protection against challenge with parental viruses. Deletion of the 9GL (B119L) gene in the highly virulent ASFV isolates Malawi Lil-20/1 (Mal) and Pretoriuskop/96/4 (Δ9GL viruses) resulted in complete protection when challenged with parental isolates. When similar deletions were created within the ASFV Georgia 2007 (ASFV-G) genome, attenuation was achieved but the protective and lethal doses were too similar. To enhance attenuation of ASFV-G, we deleted another gene, UK (DP96R), which was previously shown to be involved in attenuation of the ASFV E70 isolate. Here, we report the construction of a double-gene-deletion recombinant virus, ASFV-G-Δ9GL/ΔUK. When administered intramuscularly (i.m.) to swine, there was no induction of disease, even at high doses (106 HAD50). Importantly, animals infected with 104 50% hemadsorbing doses (HAD50) of ASFV-G-Δ9GL/ΔUK were protected as early as 14 days postinoculation when challenged with ASFV-G. The presence of protection correlates with the appearance of serum anti-ASFV antibodies, but not with virus-specific circulating ASFV-specific gamma interferon (IFN-γ)-producing cells. ASFV-G-Δ9GL/ΔUK is the first rationally designed experimental ASFV vaccine that protects against the highly virulent ASFV Georgia 2007 isolate as early as 2 weeks postvaccination. IMPORTANCE: Currently, there is no commercially available vaccine against African swine fever. Outbreaks of the disease are devastating to the swine industry and are caused by circulating strains of African swine fever virus. Here, we report a putative vaccine derived from a currently circulating strain but containing two deletions in two separate areas of the virus, allowing increased safety. Using this genetically modified virus, we were able to vaccinate swine and protect them from developing ASF. We were able to achieve protection from disease as early as 2 weeks after vaccination, even when the pigs were exposed to a higher than normal concentration of ASFV.

The progressive adaptation of a georgian isolate of African swine fever virus to vero cells leads to a gradual attenuation of virulence in swine corresponding to major modifications of the viral genome.

UNLABELLED: African swine fever virus (ASFV) causes a contagious and often lethal disease of feral and domestic swine. Experimental vaccines derived from naturally occurring, genetically modified, or cell culture-adapted ASFV have been evaluated, but no commercial vaccine is available to control African swine fever (ASF). We report here the genotypic and phenotypic analysis of viruses obtained at different passages during the process of adaptation of a virulent ASFV field isolate from the Republic of Georgia (ASFV-G) to grow in cultured cell lines. ASFV-G was successively passaged 110 times in Vero cells. Viruses obtained at passages 30, 60, 80, and 110 were evaluated in vitro for the ability to replicate in Vero cells and primary swine macrophages cultures and in vivo for assessing virulence in swine. Replication of ASFV-G in Vero cells increased with successive passages, corresponding to a decreased replication in primary swine macrophages cultures. In vivo, progressive loss of virus virulence was observed with increased passages in Vero cells, and complete attenuation of ASFV-G was observed at passage 110. Infection of swine with the fully attenuated virus did not confer protection against challenge with virulent parental ASFV-G. Full-length sequence analysis of each of these viruses revealed significant deletions that gradually accumulated in specific areas at the right and left variable ends of the genome. Mutations that result in amino acid substitutions and frameshift mutations were also observed, though in a rather limited number of genes. The potential importance of these genetic changes in virus adaptation/attenuation is discussed. IMPORTANCE: The main problem in controlling ASF is the lack of vaccines. Attempts to produce vaccines by adaptation of ASFV to cultured cell lines have been made. These attempts led to the production of attenuated viruses that conferred only homologous protection. Specifics regarding adaptation of these isolates to cell cultures have been insufficiently described. Details like the numbers of passages required to obtain attenuated viruses, genetic modifications introduced into the virus genomes along passages, and the extent of attenuation and induced protective efficacy are not readily available. In this study, we assessed the changes that lead to decreased growth in swine macrophages and to attenuation in swine. Loss of virulence, probably associated with limited replication in vivo, may lead to the lack of protective immunity in swine observed after challenge. This report provides valuable information that can be used to further the understanding of ASFV gene function, virus attenuation, and protection against infection.

African Swine Fever Virus Georgia Isolate Harboring Deletions of MGF360 and MGF505 Genes Is Attenuated in Swine and Confers Protection against Challenge with Virulent Parental Virus.

UNLABELLED: African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal disease of domestic pigs that has significant economic consequences for the swine industry. The control of African swine fever (ASF) has been hampered by the unavailability of vaccines. Experimental vaccines have been developed using genetically modified live attenuated ASFVs where viral genes involved in virus virulence were removed from the genome. Multigene family 360 (MGF360) and MGF505 represent a group of genes sharing partial sequence and structural identities that have been connected with ASFV host range specificity, blocking of the host innate response, and virus virulence. Here we report the construction of a recombinant virus (ASFV-G-ΔMGF) derived from the highly virulent ASFV Georgia 2007 isolate (ASFV-G) by specifically deleting six genes belonging to MGF360 or MGF505: MGF505-1R, MGF360-12L, MGF360-13L, MGF360-14L, MGF505-2R, and MGF505-3R. ASFV-G-ΔMGF replicates as efficiently in primary swine macrophage cell cultures as the parental virus. In vivo, ASFV-G-ΔMGF is completely attenuated in swine, since pigs inoculated intramuscularly (i.m.) with either 10(2) or 10(4) 50% hemadsorbing doses (HAD50) remained healthy, without signs of the disease. Importantly, when these animals were subsequently exposed to highly virulent parental ASFV-G, no signs of the disease were observed, although a proportion of these animals harbored the challenge virus. This is the first report demonstrating the role of MGF genes acting as independent determinants of ASFV virulence. Additionally, ASFV-G-ΔMGF is the first experimental vaccine reported to induce protection in pigs challenged with highly virulent and epidemiologically relevant ASFV-G. IMPORTANCE: The main problem for controlling ASF is the lack of vaccines. Studies focusing on understanding ASFV virulence led to the production of genetically modified recombinant viruses that, while attenuated, are able to confer protection in pigs challenged with homologous viruses. Here we have produced an attenuated recombinant ASFV derived from highly virulent ASFV strain Georgia (ASFV-G) lacking only six of the multigene family 360 (MGF360) and MGF505 genes (ASFV-G-ΔMGF). It is demonstrated, by first time, that deleting specific MGF genes alone can completely attenuate a highly virulent field ASFV isolate. Recombinant virus ASFV-G-ΔMGF effectively confers protection in pigs against challenge with ASFV-G when delivered once via the intramuscular (i.m.) route. The protection against ASFV-G is highly effective by 28 days postvaccination. This is the first report of an experimental vaccine that induces solid protection against virulent ASFV-G.

African Swine Fever Virus Georgia 2007 with a Deletion of Virulence-Associated Gene 9GL (B119L), when Administered at Low Doses, Leads to Virus Attenuation in Swine and Induces an Effective Protection against Homologous Challenge.

UNLABELLED: African swine fever virus (ASFV) is the etiological agent of an often lethal disease of domestic pigs. Disease control strategies have been hampered by the unavailability of vaccines against ASFV. Since its introduction in the Republic of Georgia, a highly virulent virus, ASFV Georgia 2007 (ASFV-G), has caused an epizootic that spread rapidly into Eastern European countries. Currently no vaccines are available or under development to control ASFV-G. In the past, genetically modified ASFVs harboring deletions of virulence-associated genes have proven attenuated in swine, inducing protective immunity against challenge with homologous parental viruses. Deletion of the gene 9GL (open reading frame [ORF] B119L) in highly virulent ASFV Malawi-Lil-20/1 produced an attenuated phenotype even when administered to pigs at 10(6) 50% hemadsorption doses (HAD50). Here we report the construction of a genetically modified ASFV-G strain (ASFV-G-Δ9GLv) harboring a deletion of the 9GL (B119L) gene. Like Malawi-Lil-20/1-Δ9GL, ASFV-G-Δ9GL showed limited replication in primary swine macrophages. However, intramuscular inoculation of swine with 10(4) HAD50 of ASFV-G-Δ9GL produced a virulent phenotype that, unlike Malawi-Lil-20/1-Δ9GL, induced a lethal disease in swine like parental ASFV-G. Interestingly, lower doses (10(2) to 10(3) HAD50) of ASFV-G-Δ9GL did not induce a virulent phenotype in swine and when challenged protected pigs against disease. A dose of 10(2) HAD50 of ASFV-G-Δ9GLv conferred partial protection when pigs were challenged at either 21 or 28 days postinfection (dpi). An ASFV-G-Δ9GL HAD50 of 10(3) conferred partial and complete protection at 21 and 28 dpi, respectively. The information provided here adds to our recent report on the first attempts toward experimental vaccines against ASFV-G. IMPORTANCE: The main problem for controlling ASF is the lack of vaccines. Studies on ASFV virulence lead to the production of genetically modified attenuated viruses that induce protection in pigs but only against homologous virus challenges. Here we produced a recombinant ASFV lacking virulence-associated gene 9GL in an attempt to produce a vaccine against virulent ASFV-G, a highly virulent virus isolate detected in the Caucasus region in 2007 and now spreading though the Caucasus region and Eastern Europe. Deletion of 9GL, unlike with other ASFV isolates, did not attenuate completely ASFV-G. However, when delivered once at low dosages, recombinant ASFV-G-Δ9GL induces protection in swine against parental ASFV-G. The protection against ASFV-G is highly effective after 28 days postvaccination, whereas at 21 days postvaccination, animals survived the lethal challenge but showed signs of ASF. Here we report the design and development of an experimental vaccine that induces protection against virulent ASFV-G.

Suburban Population of Bobcats (Lynx rufus) in Connecticut, USA, Tested Negative for SARS-CoV-2, November 2021-February 2022.

A SARS-CoV-2 genomic and serologic survey was performed in a population of bobcats (Lynx rufus) inhabiting the state of Connecticut, USA. Wild animal populations are becoming established in densely populated cities with increased likelihood of direct or indirect contact with humans, as well as with household cats and dogs. Wild-caught bobcats (n=38) tested negative for SARS-CoV-2 genomic RNA by reverse-transcription quantitative PCR and for virus-neutralizing antibodies by ELISA, suggesting that either the species is not susceptible to SARS-CoV-2 or that the surveyed population has not yet been exposed to a source of infectious virus. However, this limited survey cannot rule out that human-to-bobcat or unknown reservoir-to-bobcat transmission of the virus occurs in nature.

The complete coding sequence of a rabies lyssavirus (RABV) detected in an American black bear (Ursus americanus) in Connecticut, USA.

The complete coding sequence of a rabies lyssavirus (RABV) detected in a black bear (Ursus americanus) was generated. RNA extracted from brain tissues was amplified using reverse transcription followed by tiling PCR sequencing to obtain RABV whole viral genome. Sequencing was performed using an Illumina ISeq 100 instrument.

Classical swine fever virus p7 protein is a viroporin involved in virulence in swine.

The nonstructural protein p7 of classical swine fever virus (CSFV) is a small hydrophobic polypeptide with an apparent molecular mass of 6 to 7 kDa. The protein contains two hydrophobic stretches of amino acids interrupted by a short charged segment that are predicted to form transmembrane helices and a cytosolic loop, respectively. Using reverse genetics, partial in-frame deletions of p7 were deleterious for virus growth, demonstrating that CSFV p7 function is critical for virus production in cell cultures. A panel of recombinant mutant CSFVs was created using alanine scanning mutagenesis of the p7 gene harboring sequential three- to six-amino-acid residue substitutions spanning the entire protein. These recombinant viruses allowed the identification of the regions within p7 that are critical for virus production in vitro. In vivo, some of these viruses were partially or completely attenuated in swine relative to the highly virulent parental CSFV Brescia strain, indicating a significant role of p7 in CSFV virulence. Structure-function analyses in model membranes emulating the endoplasmic reticulum lipid composition confirmed that CSFV p7 is a pore-forming protein, and that pore-forming activity resides in the C-terminal transmembrane helix. Therefore, p7 is a viroporin which is clearly involved in the process of CSFV virulence in swine.

Whole genome sequencing and phylogenetic analysis of West Nile viruses from animals in New England, United States, 2021.

West Nile virus is a mosquito-borne Flavivirus which is the leading cause of global arboviral encephalitis. We sequenced WNVs from an American crow found in Connecticut and an alpaca found in Massachusetts which were submitted to the Connecticut Veterinary Medical Diagnostic Laboratory (CVMDL). We report here the complete protein-coding sequences (CDS) of the WNVs (WNV 21-3957/USA CT/Crow/2021 and WNV 21-3782/USA MA/Alpaca/2021) and their phylogenetic relationship with other WNVs recovered from across the United States. In the phylogenetic analysis, the WNVs from this study belonged to the WNV lineage 1. The WNV 21-3957/USA CT/Crow/2021 clustered with WNVs from a mosquito and birds in New York during 2007-2013. Interestingly, the virus detected in the alpaca, WNV 21-3782/USA MA/Alpaca/2021 clustered with WNVs from mosquitos in New York, Texas, and Arizona during 2012-2016. The genetic differences between the viruses detected during the same season in an American crow and an alpaca suggest that vector-host feeding preferences are most likely driving viral transmission. The CDS of the WNVs and their phylogenetic relationships with other WNVs established in this study would be useful as reference data for future investigations on WNVs. Seasonal surveillance of WNV in birds and mammals and the genetic characterization of detected viruses are necessary to monitor patterns of disease presentations and viral evolution within a geographical area.

Whole genome sequencing and phylogenetic analysis of African swine fever virus detected in a backyard pig in Mongolia, 2019.

African swine fever (ASF) is a highly contagious and fatal disease affecting domestic and wild pigs caused by the African swine fever virus (ASFV). Since the first outbreak in China in August 2018, ASF has spread rapidly in Asia. and the first case in Mongolia was confirmed in January 2019. In this study, we report the first whole genome sequence of an ASFV (ASFV SS-3/Mongolia/2019) detected from a backyard pig in Mongolia in February 2019 using whole genome sequencing. We analyzed their phylogenetic relationship with other genotype II ASFVs from Eurasia. The ASFV SS-3/Mongolia/2019 belonged to genotype II (p72 and p54), serogroup 8 (CD2v), Tet-10a variant (pB602L), and IGRIII variant (intergenic region between the I73R/I329L genes). A total of five amino acid substitutions were observed in MGF 360-10L, MGF 505-4R, MGF 505-9R, NP419L, and I267L genes compared to the ASFV Georgia 2007/1 virus. ML phylogenetic analysis of the whole genome sequence showed that the virus shares a high nucleotide sequence identity with ASFVs recently identified in Eastern Europe and Asia and clustered with the ASFV/Zabaykali/WB5314/2020|Russia|2020 virus which was identified at the border between the Russian Federation and Mongolia in 2020. Our results suggest that trans boundary spread of ASF occurred through close geographic proximity.

Sulawesi tortoise adenovirus-1 in two impressed tortoises (Manouria impressa) and a Burmese star tortoise (Geochelone platynota).

Sulawesi tortoise adenovirus-1 (STAdV-1) is a newly discovered virus infecting endangered and threatened tortoises. It was initially described from a confiscated group of 105 Sulawesi tortoises (Indotestudo forsteni) obtained by the Turtle Survival Alliance and distributed to five sites with available veterinary care across the United States. In a 3-yr period from the initial outbreak, one multi-species collection that rehabilitated and housed adenovirus-infected Sulawesi tortoises experienced deaths in impressed tortoises (Manouria impressa) and a Burmese star tortoise (Geochelone platynota). Impressed tortoises that died had evidence of systemic viral infection with histopathologic features of adenovirus. Adenovirus was identified by consensus nested polymerase chain reaction (PCR) testing and subsequent sequencing of PCR products. Sequencing indicated that the adenovirus infecting these impressed tortoises and Burmese star tortoise was STAdV-1. In one impressed tortoise, viral infection was confirmed using transmission electron microscopy. In situ hybridization using a semiautomated protocol and fluorescein-labeled riboprobe identified STAdV-1 inclusions in spleen, liver, kidney, and testis of one impressed tortoise. The impact of this virus on captive and wild populations of tortoises is unknown; however, these findings indicate that STAdV-1 can be transmitted to and can infect other tortoise species, the impressed tortoise and Burmese star tortoise, when cohabitated with infected Sulawesi tortoises.

Genomic Features of Salmonella enterica Subspecies houtenae Serotype 45:g,z51:- Isolated from Multiple Abdominal Abscesses of an African Fat-Tailed Gecko, United States, 2020.

Salmonella enterica subsp. houtenae (S. houtenae) is a common subspecies in reptiles and has been implicated as a source of serious and life-threatening diseases in humans. Although occurrence and significance of S. houtenae infections have been extensively studied, the genetic features of S. houtenae have remained unknown due to a lack of available high-quality genome sequences. We obtained the complete genome sequence of S. houtenae 45:g,z51:- strain 20-369 isolated from multiple abdominal abscesses of an African fat-tailed gecko (Hemitheconyx caudicinctus) using Nanopore and Illumina sequencing technologies and generated the 4.65Mbp complete genome sequence of the S. houtenae str. 20-369. We annotated and analyzed the genome sequence with the aim to gain a deeper understanding of the genome characteristics associated with its pathogenicity. Overall, this study found several interesting genomic features such as pseudogene formation, virulence gene profile, and novel genomic islands. This study provides basis for an understanding possible genetic mechanism underlying pathogenicity of S. houtenae 45:g,z51:- as well as a high-quality genome reference for future comparison studies.

Complete mitochondrial genome of Asian longhorned tick, Haemaphysalis longicornis, Neumann, 1901 (Acari: Ixodida: Ixodidae) identified in the United States.

Haemaphysalis longicornis (Ixodida: Ixodidae), the Asian longhorned tick, which is native to temperate East Asia, has been recently detected in the northeastern region of the United States, drawing concerns about its potential impact on the US animal and public health sectors. Knowledge about the genetic features of H. longicornis found in the US is limited. Therefore, we sequenced the complete mitochondrial genome (mt-genome) from two H. longicornis ticks recently collected in the State of New York, USA, in 2020. These ticks were morphologically identified and tested for tick-borne pathogens at the Connecticut Veterinary Medical Diagnostic Laboratory (Storrs, CT). The mt-genome was 14,694 bp in length and encoded 37 genes, including 13 protein-coding genes, 22 transfer RNAs, and two ribosomal RNAs. Phylogenetic analysis showed that the mt-genome clustered with those of other H. longicornis identified in China. The mt-genome sequence was 99.7% identical to a H. longicornis mt-genome (GenBank: MK439888) collected in China. The cox1 gene haplotype in these ticks belonged to the H1 type, which is the dominant haplotype present in central NJ and Staten Island, NY. The complete mt-genome data are needed to provide insights into genetic changes and phylogenetic studies of H. longicornis ticks.

Genetic features of Salmonella enterica subspecies diarizonae serovar 61:k:1,5 isolated from abortion cases in sheep, United States, 2020.

Salmonella enterica subspecies diarizonae serovar 61:(k):1, 5, (7) (sheep associated S. diarizonae, SASd) is the most common Salmonella serotype identified in sheep flocks. Despite the involvement with animal and human infections, there is limited information regarding virulence profiles of SASds and their antibiotic resistance gene complement, particularly for those circulating in the U.S. In this study, we genetically characterized three SASds, 20-265, 20-269, and 20-312, isolated from sheep placental tissues during an abortion storm affecting a flock in Connecticut during 2020. SASds were the only bacteria isolated from analyzed sheep tissues. The isolates were sensitive to all the antibiotics tested, but all these SASd isolates carry the aminoglycoside resistance gene, aac(6')-Iaa, and a chromosomal substitution in the parC gene. The proportion of pseudogenes (5.3-5.5%) was similar among the isolates, and these SASds carry IncX1 type plasmids. Comparing with the SASds isolates from Enterobase, the three isolates showed an identical genomic virulence profile carrying virulence genes in the conserved set of other SASd isolates except for steC, iagB, iacP, sseI, and slrP genes. In the SNP-based phylogenetic analysis, SASd sequences were grouped into group A-C, and the group C was further subdivided into subgroup C1-C6. The three isolates clustered with other SASd isolates from the U.S. and Canada in subgroup C6. SASd isolates in the identical phylogenetic groups tended to have similar geographical origin. The results of our study did not provide conclusive evidence about which are the genetic traits that trigger SASds to become virulent in sheep, but our data will provide a point for comparative studies of this Salmonella serovar.

Whole Genome Sequencing and Phylogenetic Analysis of Rabies Viruses from Bats in Connecticut, USA, 2018-2019.

We performed whole genome sequencing and genetic characterization of rabies viruses (RABV) detected in bats submitted to the Connecticut Veterinary Medical Diagnostic Laboratory (CVMDL) during 2018-2019. Among 88 bats submitted to CVMDL, six brain samples (6.8%, 95% confidence interval: 1.6% to 12.1%) tested positive by direct fluorescent antibody test. RABVs were detected in big brown bats (Eptesicus fuscus, n = 4), a hoary bat (Lasiurus cinereus, n = 1), and an unidentified bat species (n = 1). Complete coding sequences of four out of six detected RABVs were obtained. In phylogenetic analysis, the RABVs (18-62, 18-4347, and 19-2274) from big brown bats belong to the bats EF-E1 clade, clustering with RABVs detected from the same bat species in Pennsylvania and New Jersey. The bat RABV (19-2898) detected from the migratory hoary bat belongs to the bats LC clade, clustering with the eleven viruses detected from the same species in Arizona, Washington, Idaho, and Tennessee. The approach used in this study generated novel data regarding genetic relationships of RABV variants, including their reservoirs, and their spatial origin and it would be useful as reference data for future investigations on RABV in North America. Continued surveillance and genome sequencing of bat RABV would be needed to monitor virus evolution and transmission, and to assess the emergence of genetic mutations that may be relevant for public health.

Deletion of CD2-like gene from the genome of African swine fever virus strain Georgia does not attenuate virulence in swine.

The CD2-like African swine fever virus (ASFV) gene 8DR, (also known as EP402R) encodes for a structural transmembrane glycoprotein that has been shown to mediate hemadsorption and be involved in host immunomodulation as well as the induction of protective immune response. In addition, several natural ASFV isolates showing decreased virulence in swine has been shown to be non-hemadsorbing suggesting an association between altered or deleted forms of 8DR and virus attenuation. Here we demonstrate that deletion of 8DR gene from the genome of ASFV Georgia2010 isolate (ASFV-G-Δ8DR) does not significantly alter the virulence of the virus. ASFV-G-Δ8DR inoculated intramuscularly or intranasally (in a range of 102 to 104 TCID50) produced a clinical disease in domestic pigs indistinguishable from that induced by the same doses of the virulent parental ASFV Georgia2010 isolate. In addition, viremia values in ASFV-G-Δ8DR do not differ from those detected in animals infected with parental virus. Therefore, deletion of 8DR gene is not associated with a noticeable decrease in virulence of the ASFV Georgia isolate.

Actinomyces hyovaginalis-associated lymphadenitis in a Nubian goat.

A 6-year-old Nubian goat with a history of progressive weight loss and cough was presented for necropsy. The goat tested negative for antibodies to caseous lymphadenitis and caprine arthritis and encephalitis by hemagglutination inhibition assay and enzyme-linked immunosorbent assay, respectively. Postmortem examination revealed marked enlargement and, with histopathology, a fibrinopurulent necrotizing lymphadenitis of a tracheobronchial lymph node, with an appearance similar to that reported in cases of caseous lymphadenitis. An organism characterized by molecular methods as Actinomyces hyovaginalis was isolated together with Staphylococcus spp. and Streptococcus spp. from the lesion. No Corynebacterium pseudotuberculosis was recovered. To the authors' knowledge, this is the first isolation of A. hyovaginalis from a goat. Although the exact contribution of A. hyovaginalis to the lesion remains to be established, this case demonstrates that A. hyovaginalis should be considered in cases of caseous lymphadenitis-type lesions, especially when C. pseudotuberculosis has been excluded.

Systemic iridovirus infection in the Banggai cardinalfish (Pterapogon kauderni Koumans 1933).

Iridoviruses infect food and ornamental fish species from a wide range of freshwater to marine habitats across the globe. The objective of the current study was to characterize an iridovirus causing systemic infection of wild-caught Pterapogon kauderni Koumans 1933 (Banggai cardinalfish). Freshly frozen and fixed specimens were processed for histopathologic evaluation, transmission electron microscopic examination, virus culture, molecular virologic testing, microbiology, and in situ hybridization (ISH) using riboprobes. Basophilic granular cytoplasmic inclusions were identified in cytomegalic cells often found beneath endothelium, and hexagonal virus particles typical of iridovirus were identified in the cytoplasm of enlarged cells by transmission electron microscopy. Attempts at virus isolation in cell culture were unsuccessful; however, polymerase chain reaction (PCR)-based molecular testing resulted in amplification and sequencing of regions of the DNA polymerase and major capsid protein genes, along with the full-length ATPase gene of the putative iridovirus. Virus gene sequences were then used to infer phylogenetic relationships of the P. kauderni agent to other known systemic iridoviruses from fishes. Riboprobes, which were transcribed from a cloned PCR amplification product from the viral genome generated hybridization signals from inclusions within cytomegalic cells in histologic sections tested in ISH experiments. To the authors' knowledge, this is the first report of a systemic iridovirus from P. kauderni. The pathologic changes induced and the genomic sequence data confirm placement of the Banggai cardinalfish iridovirus in the genus Megalocytivirus family Iridoviridae. The ISH provides an additional molecular diagnostic technique for confirmation of presumptive infections detected in histologic sections from infected fish.

Systemic adenovirus infection in Sulawesi tortoises (Indotestudo forsteni) caused by a novel siadenovirus.

A novel siadenovirus was identified in the Sulawesi tortoise (Indotestudo forsteni). A group of 105 Sulawesi tortoises was obtained by the Turtle Survival Alliance. Many of the tortoises were in poor health. Clinical signs included anorexia, lethargy, mucosal ulcerations and palatine erosions of the oral cavity, nasal and ocular discharge, and diarrhea. Initial diagnostic tests included fecal testing for parasites, complete blood count and plasma biochemical analysis, mycoplasma serology, and polymerase chain reaction (PCR) testing for intranuclear coccidia and chelonian herpesvirus. Treatment included administration of antibiotics, antiparasitic medications, parenteral fluids, and nutritional support. Tissue samples from animals that died were submitted for histopathologic evaluation. Histopathologic examination revealed systemic inflammation and necrosis associated with intranuclear inclusions consistent with a systemic viral infection in 35 tortoises out of 50 examined. Fecal testing results and histopathologic findings revealed intestinal and hepatic amoebiasis and nematodiasis in 31 animals. Two of 5 tortoises tested by PCR were positive for Chlamydophila sp. Aeromonas hydrophila and Escherichia coli were cultured from multiple organs of 2 animals. The mycoplasma serology and PCR results for intranuclear coccidia and chelonian herpesvirus were negative. Polymerase chain reaction testing of tissues, plasma, and choanal/cloacal samples from 41 out of 42 tortoises tested were positive for an adenovirus, which was characterized by sequence analysis and molecular phylogenetic inference as a novel adenovirus of the genus Siadenovirus. The present report details the clinical and anatomic pathologic findings associated with systemic infection of Sulawesi tortoises by this novel Siadenovirus, which extends the known reptilian adenoviruses to the chelonians and extends the known genera of reptilian Adenoviridae beyond Atadenovirus to include the genus Siadenovirus.

Patterns of cellular gene expression in swine macrophages infected with highly virulent classical swine fever virus strain Brescia.

Experimental exposure of swine to highly virulent classical swine fever virus (CSFV) strain Brescia causes an invariably fatal disease of all infected animals by 8-14 days post-infection. Host mechanisms involved in this severe outcome of infection have not been clearly established. To understand these mechanisms, we analyzed the response of primary cultured swine macrophages, a CSFV primary target cell, to infection with Brescia strain. Steady state levels of mRNA accumulation were assessed for 58 genes involved in modulation of the host immune response, at 24 and 48 h post-infection (hpi), by means of quantitative reverse transcription real-time PCR analysis (qrt-PCR). Eighteen genes showed altered expression upon infection with CSFV strain Brescia including: cytokines (IL-1alpha, IL-1beta, IL-6, and IL-12p35); cytokine receptors (IL-2Ralpha, IL-12Rbeta, and TGF-betaIIIR); chemokines (IL-8, AMCF-1, AMCF-2, MCP-2, and RANTES); interferons (INFalpha and INFbeta); and toll-like receptors (TLR3, TLR5, TLR9, and TLR10). Although these genes are associated with mechanisms of innate immune response and antiviral activity, their altered expression does not curtail CSFV Brescia growth kinetics and virus yield in swine macrophages. Data gathered here suggests that the observed gene expression profile might explain immunological and pathological changes associated with virulent CSFV infections.