Failure to establish chronic infection of the reproductive tract of the male horse with a South African asinine strain of equine arteritis virus (EAV)

Eight sexually mature horse stallions were inoculated intranasally with a South African asinine strain of EAV, a strain that was isolated from the semen of a donkey carrier. All horses developed fever, with maximum rectal temperatures of 38,9-39,9°C recorded 3-6 d post challenge. Six horses showed very mild clinical signs of equine viral arteritis and two were asymptomatic. The virus was recovered from the nasopharynxes of six horses 2-7 d after inoculation, and from buffy-coat samples of all horses, 2- 11 d after inoculation. Seroconversion to EAV was detected on days 8 and 10 and peak serum-virusneutralizing antibody titres ranging from log₁₀ 1,2 - 1,8, on days 14-20 after challege. The titres varied from log₁₀ 0,9 - 1,2 after about 10 weeks, when the experiment was terminated. In three stallions euthanased on days 5, 7 and 9 after challenge, virus was detected inconsistently in different parts of the reproductive tract and urine. No virus was isolated from the tissues of the reproductive tract collected from stallions on days 16, 23 and 68 after challenge. Five stallions were bred to six seronegative mares between 13 and 34 d post challenge. No clinical signs of EAV were observed, and neither was seroconversion detected in any of the mares after mating. No virus was recovered from semen samples collected at the time of breeding. The results of this study demonstrated that the tissues of the reproductive tracts of the stallions did not become persistently infected with a South African asinine strain of EAV.The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. Adobe Acrobat X Pro was used to OCR the text and also for the merging and conversion to the final presentation PDF-format

Effect of the South African asinine-94 strain of equine arteritis virus (EAV) in pregnant donkey mares and duration of maternal immunity in foals

Clinical, virological and serological responses were investigated in five pregnant donkey mares after experimental exposure to the South African asinine-94 strain of equine arteritis virus (EAV), and the duration of maternal immunity to EAV was studied in their foals. In four intranasally inoculated mares, fever with maximum rectal temperatures of 39,1-40,7°C was recorded 2-11 d after challenge. All the inoculated mares developed mild depression, and a serous ocular and nasal discharge; in three mares mild conjunctivitis was observed. The virus was recovered from the nasopharynx and from buffy-coat samples of all the mares 3-10 d, and 2-16 d post inoculation (p.i.), respectively. Seroconversion to EAV was detected on days 8- 10 p.i. Peak serum-virus- neutralizing antibody titres of log₁₀1,8-2,4, and lgG ELISA OD values of 0,85-2,15 were recorded 2-3 weeks p.i. The in-contact (p.c.) control mare developed fever on days 15-19 post exposure, and showed mild clinical signs of equine viral arteritis similar to those observed in the inoculated mares. Seroconversion to EAV was detected in the p.c. mare on day 20 post exposure, and virus was isolated from nasal swabs and blood samples collected at the time of the febrile response and 1-3 d afterwards. None of the mares aborted. After they had given normal birth 45-128 d p.i. or after p.c. exposure, no virus could be isolated from their placentas. The concentration of EAV-neutralizing antibody in colostrum was two to eight times higher than in serum samples collected at the time of parturition. All the foals born to infected mares were clinically normal at the time of birth and throughout the subsequent 1-2 months of observation. No EAV was recovered from the bully-coat fraction of blood samples collected at birth nor from those collected on days 1, 2 and 7 after birth. Also, no virus-serum- neutralizing or lgG ELISA antibody to EAV was detected in sera collected immediately after birth before the foals started nursing. The colostrum-derived maternal antibodies against EAV gradually declined and could not be detected by either the VN test or ELISA for 2-3 months after birth. This study demonstrates that the asinine-94 strain of EAV does not cause abortion in pregnant donkey mares. Furthermore, no carrier state could be demonstrated in foals born to mares infected at the time of pregnancy.The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. Adobe Acrobat X Pro was used to OCR the text and also for the merging and conversion to the final presentation PDF-format

Serological evidence of equine arteritis virus in donkeys in South Africa

This paper reports the first serological evidence of exposure of donkeys to equine arteritis virus. Seven hundred and thirty-four serum samples collected between 1989 and 1992 from donkeys in different areas of South Africa were examined for the presence of antibodies against this virus by a microneutralization test Seventeen percent of serum samples tested positive. The distribution of seropositive animals varied from none in the western Cape Province and the Transvaal Highveld to 30% in the northern Transvaal. The country-wide distribution of serologically positive donkeys suggests a longstanding presence of the virus in South Africa.The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. Adobe Acrobat XI Pro was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.mn201

Prevalence of antibodies against some equine viruses in zebra (Zebra burchelli) in the Kruger National Park, 1991-1992

The presence of antibodies against equine encephalosis virus (EEV) and equid herpesvirus 1 and 4 in zebra in the Kruger National Park (KNP) was demonstrated. The ability of zebra to maintain immunity against EEV is illustrated by the appearance of neutralizing antibodies in most zebra foals within months of losing their maternal immunity. This occurs in every month of the year, even in winter. The high proportion of serologically positive foals in winter is ascribed to the presence of large numbers of susceptible foals and sufficient numbers of Culicoides vectors even at that time of the year. The high prevalence of antibodies against both herpesviruses is similar to the situation in horses and suggests that herpesvirus infection is endemic among zebra in the KNP. No evidence of infection with either A/equine/H3N8 or equine arteritisvirus could be found.The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. Adobe Acrobat XI Pro was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.mn201

The use of sucrose-acetone-extracted Rift Valley fever virus antigen derived from cell culture in an indirect enzyme-linked immunosorbent assay and haemagglutination-inhibition test

A sucrose-acetone-extracted, Madin-Darby-bovine-kidney (MDBK)-derived Rift Valley fever virus (RVFV) antigen was tested both in an indirect ELISA and a haemagglutination-inhibition test for its ability to detect serum antibodies to RVFV. Optimal conditions for antigen concentration, serum and conjugate dilutions for the ELISA were established by checkerboard titration. The specificity and sensitivity of ELISA were determined by the use of paired pre- and post-vaccination sheep-serum samples. Compared with the virus neutralization test, the overall ELISA specificity and sensitivity were 97,4 and 97,3 %, respectively. There was a 100% correlation between the results obtained in haemagglutination-inhibition tests with a RVFV sucrose-acetone-extracted antigen derived from hamster liver, and from MDBK cells. A total of 10 582 field-serum samples (84 cattle, 3 659 sheep, 6 839 goats) collected in 1994-1995 from animals of unknown vaccination status in different regions of South Africa were tested with ELISA for antibodies against RVFV. There were no seropositive cattle, 0,16% seropositive sheep and 0,12% seropositive goats. This study demonstrates the potential diagnostic application of cell-culture-derived, sucrose-acetone-extracted RVFV antigen in an indirect ELISA and HI test.The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. Adobe Acrobat XI Pro was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.mn201

Transmission of the South African asinine strain of equine arteritis virus (EAV) among horses and between donkeys and horses

Lateral and sexual transmission of EAV among horses and lateral transmission between donkeys and horses were attempted by experimental infection with the South African asinine strain. Clinical, immunological and virological responses were evaluated. All intramuscularly inoculated horses developed very mild clinical signs, were viraemic, shed virus from nasopharynx, and seroconverted. Lateral infection was demonstrated in one in-contact mare. Reinfection of two stallions by intranasal instillation was shown by virus recovery from bully-coat cultures. After nasal instillation of virus, one stallion which did not become infected by in-contact exposure, showed slight serous nasal and ocular discharge, contained virus in a blood and nasopharynx and seroconverted. Attempts to transmit the virus from seropositive stallions to seronegative mares by breeding, were not successful; no virus was isolated from semen. All inoculated donkeys and three in-contact horses showed clinical signs consistent with an EAV infection. Although virus was isolated from donkey buffy-coat preparations and the nasopharynx, and they seroconverted, no virus was isolated from the horses, and they failed to seroconvert; it was assumed that their clinical signs were due to factors unrelated to EAV. The South African strain of EAV appears to be poorly transmissible to horses, supporting the findings of other field studies which indicate a widespread distribution and long-standing presence of the virus among South African donkeys, but a very restricted prevalence of seropositive horses.The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. Adobe Acrobat X Pro was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.mn201

Next-generation sequencing of southern African Crimean-Congo haemorrhagic fever virus isolates reveals a high frequency of M segment reassortment

Crimean Congo haemorrhagic fever virus (CCHFV) is a bunyavirus with a single-stranded RNA genome consisting of three segments (S, M, L), coding for the nucleocapsid protein, envelope glycoproteins and RNA polymerase, respectively. To date only five complete genome sequences are available from southern African isolates. Complete genome sequences were generated for 10 southern African CCHFV isolates using next-generation sequencing techniques. The maximum-likelihood method was used to generate tree topologies for 15 southern African plus 26 geographically distinct complete sequences from GenBank. M segment reassortment was identified in 10/15 southern African isolates by incongruencies in grouping compared to the S and L segments. These reassortant M segments cluster with isolates from Asia/Middle East, while the S and L segments cluster with strains from South/West Africa. The CCHFV M segment shows a high level of genetic diversity, while the S and L segments appear to co-evolve. The reason for the high frequency of M segment reassortment is not known. It has previously been suggested that M segment reassortment results in a virus with high fitness but a clear role in increased pathogenicity has yet to be shown.The National Health Laboratory Service Research Trust, the Polio Research Foundation, South Africa, and University of the Free State Cluster funding.http://journals.cambridge.org/action/displayJournal?jid=HYG2015-05-30am201

Antibody responses to Marburg virus in Egyptian rousette bats and their role in protection against infection

Egyptian rousette bats (ERBs) are reservoir hosts for the Marburg virus (MARV). The immune dynamics and responses to MARV infection in ERBs are poorly understood, and limited information exists on the role of antibodies in protection of ERBs against MARV infection. Here, we determine the duration of maternal immunity to MARV in juvenile ERBs, and evaluate the duration of the antibody response to MARV in bats naturally or experimentally infected with the virus. We further explore whether antibodies in previously naturally exposed bats is fully protective against experimental reinfection with MARV. Maternal immunity was lost in juvenile ERBs by 5 months of age. Antibodies to MARV remained detectable in 67% of experimentally infected bats approximately 4 months post inoculation (p.i.), while antibodies to MARV remained present in 84% of naturally exposed bats at least 11 months after capture. Reinfection of seropositive ERBs with MARV produced an anamnestic response from day 5 p.i. Although PCR-defined viremia was present in 73.3% of reinfected ERBs, replicating virus was recovered from the serum of only one bat on day 3 p.i. The negative PCR results in the salivary glands, intestines, bladders and reproductive tracts of reinfected bats, and the apparent absence of MARV in the majority of swabs collected from these bats suggest that reinfection may only play a minor role in the transmission and maintenance of MARV amongst ERBs in nature.The National Institute for Communicable Diseases, the National Research Foundation of South Africa (86228), the Poliomyelitis Research Foundation (13/54), and the University of Pretoria (postgraduate bursary).http://www.mdpi.com/journal/virusesam2018Medical VirologyMicrobiology and Plant Patholog

Comparative analysis of the L, M, and S RNA segments of Crimean-Congo haemorrhagic fever virus isolates from southern Africa

Crimean–Congo haemorrhagic fever virus (CCHFV) is a member of the Bunyaviridae family with a tripartite, negative sense RNA genome. This study used predictive software to analyse the L (large), M (medium), and S (small) segments of 14 southern African CCHFV isolates. The OTU-like cysteine protease domain and the RdRp domain of the L segment are highly conserved among southern African CCHFV isolates. The M segment encodes the structural glycoproteins, GN and GC, and the non-structural glycoproteins which are post-translationally cleaved at highly conserved furin and subtilase SKI-1 cleavage sites. All of the sites previously identified were shown to be conserved among southern African CCHFV isolates. The heavily O-glycosylated N-terminal variable mucin-like domain of the M segment shows the highest sequence variability of the CCHFV proteins. Five transmembrane domains are predicted in the M segment polyprotein resulting in three regions internal to and three regions external to the membrane across the GN, NSM and GC glycoproteins. The corroboration of conserved genome domains and sequence identity among geographically diverse isolates may assist in the identification of protein function and pathogenic mechanisms, as well as the identification of potential targets for antiviral therapy and vaccine design. As detailed functional studies are lacking for many of the CCHFV proteins, identification of functional domains by prediction of protein structure, and identification of amino acid level similarity to functionally characterised proteins of related viruses or viruses with similar pathogenic mechanisms are a necessary step for selection of areas for further study.National Health Laboratory Service Research Trust, the Polio Research Foundation, South Africa and University of the Free State Cluster funding.http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1096-90712016-05-31hb201

Synchronized shift of oral, faecal and urinary microbiotas in bats and natural infection dynamics during seasonal reproduction

Seasonal reproduction is a period of extreme physiological and behavioural changes, yet we know little about how it may affect host microbial communities (i.e. microbiota) and pathogen transmission. Here, we investigated shifts of the bacterial microbiota in saliva, urine and faeces during the seasonal reproduction of bats in South Africa, and test for an interaction in shedding patterns of both bacterial (Leptospira) and viral (adeno- and herpesviruses) agents. Based on a comparative approach in two cave-dwelling bat species and high-throughput sequencing of the 16S rRNA gene, we demonstrated a clear signature in microbiota changes over the reproduction season, consistent across the multiple body habitats investigated, and associated with the sex, age and reproductive condition of bats. We observed in parallel highly dynamic shedding patterns for both bacteria and viruses, but did not find a significant association between viral shedding and bacterial microbiota composition. Indeed, only Leptospira shedding was associated with alterations in both the diversity and composition of the urinary microbiota. These results illustrate how seasonal reproduction in bats substantially affects microbiota composition and infection dynamics, and have broad implications for the understanding of disease ecology in important reservoir hosts, such as bats.In part by the National Research Foundation (NRF) of South Africa: the NRF-DST South African Research Chair held by Prof Markotter, grant no. 98339, as well as grant numbers 92524, 85756 and 91496, and grant UID 78566 (NRF RISP grant for the ABI3500). This research was partially supported by the Cooperative Agreement Number [5 NU2GGH001874-02-00], funded by the Centers for Disease Control and Prevention, USA. M.D.’s postdoctoral fellowship was funded by a Capacity Building Grant from the National Research Foundation, South Africa (grant no. UID 92524).http://rsos.royalsocietypublishing.orgam2018Centre for Wildlife ManagementMammal Research InstituteMedical Virolog