Evaluation of the effectiveness of foot-and-mouth disease vaccination of animals in the buffer zone of the Republic of Armenia in 2016-2020.

BACKGROUND: Foot-and-mouth disease (FMD) is a high impact viral disease of livestock for which vaccines are extensively used for limiting the spread of infection. Armenia shares a border with both Turkey and Iran where FMD is endemic, making vaccination an important component of Armenia's control strategy. Additionally, Armenian veterinary services utilize both passive and active monitoring for prevention control. METHODS: We sought to determine the immune status of animals vaccinated against FMD and to evaluate the effectiveness of our vaccination policy in Armenia. This was conducted in three regions including Shirak, Armavir, and Ararat Region which are located in the buffer zones that border Turkey and Iran. Through active monitoring in 2020, we studied blood serum samples from cattle and sheep using an enzyme immunoassay to determine the level of immune animals in these regions following the use of a polyvalent inactivated vaccine containing FMDV serotypes A, O, and Asia-1 that are relevant for this region. ELISA titers were assessed at 28, 90, and 180 days after vaccination in cattle of three age groups at the time of initial vaccination: 4-6 months, 6-18 months and ≥ 24 months of age with sheep of all ages. RESULTS: The 3 age groups of cattle had similarly high levels of immunity with over 90% of the cattle showing a ≥ 50% protective titer 28 days after the first vaccination. By day 90, titers in cattle from the initial 4-18-month age groups dropped below 58% across the 3 serotypes and at or below 80% for the oldest cattle ≥ 24 months. Re-vaccination of cattle at 120 days did improve protective titers but never reached the level of immunity of the first vaccination. Sheep showed a similar rapid drop to less than 50% having a ≥ 50% protective titer at 90 days emphasizing the need for continual revaccination. CONCLUSIONS: The results of this study have important implications for the current FMD vaccine policy in Armenia and improves our understanding of the rapid loss of protective titers over short periods. Since small ruminants are only vaccinated once per year and vaccination titers drop rapidly by 90 days suggests that they are vulnerable to FMD and that vaccination protocols need to be updated. Cattle should continue to be vaccinated every 3-6 months depending on their age to maintain a protective level of antibodies to protect them from FMD. More studies are needed to understand the possible role of small ruminants in the epidemiology of FMD and to evaluate revaccination at shorter intervals. These results show the concerns of rapid loss of protection to both cattle and small ruminants following 1 or more doses of commercial vaccines and that additional vaccines need to be evaluated in both groups to know how often they must be vaccinated to provide full protection. The addition of challenge studies should also be considered to better understand the level of protection as measured by serology and how it relates to protection from challenge. These results should be considered by anyone using these vaccines in cattle and sheep at longer than 3 month intervals.

The Serological Response in Cattle following Administration of a Heterologous Sheep Pox Virus Strain Vaccine for Protection from Lumpy Skin Disease; Current Situation in Armenia.

Lumpy skin disease (LSD) is a highly infectious viral disease of cattle caused by LSD virus (LSDV), which was first reported in Armenia in late 2015. It was identified in pasture-raised cattle near the border with Iran. Currently, vaccination plays a key role in preventing further incursion of disease in high-risk areas. The purpose of this work was to assess the quality of vaccination currently used in Armenia by determining the immune response of the heterologous dry culture sheep pox virus-based vaccine against LSD in cattle. Seroprevalence and seroconversion testing was carried out using an ELISA to detect specific antibodies against LSD before and 30 days after vaccination in three adjacent regions of Armenia (Ararat, Armavir, Gegharkunik). Ixodes ticks were also examined for the presence of LSDV via real-time PCR. We found that the heterologous vaccine used in Armenia creates a high level of population immunity of 86.09% (83.83-87.97%) and no adverse side effects were observed in cattle. Of the 6 types of Ixodes ticks identified and tested, we found no evidence of LSDV circulating in these vectors. These results suggest that regular serological monitoring via ELISA and heterologous vaccination should continue in areas of Armenia at high risk for incursion of LSD to reduce the spread of this highly infectious transboundary disease.

Coinfections of African swine fever virus, porcine circovirus 2 and 3, and porcine parvovirus 1 in swine in Nigeria.

As pig production increases in Africa, it is essential to identify the pathogens that are circulating in the swine population to assess pig welfare and implement targeted control measures. For this reason, DNA samples collected from pigs in Nigeria in the context of African swine fever monitoring were further screened by PCR for porcine circovirus 2 (PCV-2), porcine circovirus 3 (PCV-3), and porcine parvovirus 1 (PPV1). Forty-seven (45%) pigs were positive for two or more pathogens. Sequence analysis identified PCV-2 genotypes a, b, and d, while limited genetic heterogenicity was observed among PCV-3 strains. All except one of the PPV1 sequences were genetically distinct from those previously identified in other countries.

Molecular characterization of African swine fever viruses from Burkina Faso, 2018.

BACKGROUND: African swine fever (ASF) is a viral hemorrhagic disease of domestic and wild swine. ASF has been endemic in Burkina Faso since 2003. In October 2018, substantial pig deaths occurred in Ouagadougou and two neighboring municipalities in central Burkina Faso. Following these mortalities, the veterinary extension services carried out investigations to begin control measures and collect samples. METHODS: We performed real-time PCR for diagnostic confirmation and molecular characterization of the virus based on the partial P72, the complete p54, the partial CD2v, and partial B602L genes. RESULTS: The field study revealed that mortalities started two weeks before our investigations. The real-time PCR results confirmed ASFV DNA in twenty samples out of sixty-two blood samples collected in four different locations. The sequencing and phylogenetic analysis showed that ASFVs causing these outbreaks belong to genotype I and serogroup 4. The study of the CVR showed 4 TRS variants, and that of the CD2v amino acid sequence revealed five variants based on the number of deleted KCPPPK motifs in the C-terminal proline-reach region of the protein. CONCLUSIONS: The existence of multiple variants in these outbreaks shows the importance of molecular characterization to understand the evolution of ASFV isolates and the link between epidemics.

Symptomatic and asymptomatic cases of African swine fever in Tanzania.

African swine fever (ASF) is an acute, highly contagious and deadly viral haemorrhagic disease of domestic pigs caused by African swine fever virus (ASFV). In ASF endemic countries, there are an increasing number of reports on circulating ASFV strains with different levels of virulence causing a broad range of clinical symptoms in susceptible animals. Tanzania, where ASFV is endemic since 2001, recorded several outbreaks including symptomatic and asymptomatic cases between 2015 and 2017. We collected 35 clinical samples from four outbreaks for diagnostic confirmation and sequenced the partial B646L (p72), the full E183L (p54) gene, the central variable region of the B602L gene and the intergenic region between the I73R and I329L genes to characterize molecularly the new ASFV isolates and analyse their relatedness with previously reported Tanzanian and foreign isolates. We detected ASFV in 21 samples, 15 from symptomatic and six from asymptomatic pigs. Phylogenetic analyses based on the partial p72 gene and the complete p54 (E183L) genes revealed that the ASFVs in samples from symptomatic pigs belonged to genotypes II and those in samples from asymptomatic pigs belonged to genotype IX. The CVR profiles of the p72 genotype II and genotype IX isolates differed between each other and from previously published Tanzanian sequences. The sequence analysis of the intergenic region between the I73R and I329L for the 2017 genotype II isolates showed the absence of one GGAATATATA motif in those isolates. This study showed the simultaneous circulation of two different ASFV genotypes with different levels of pathogenicity in Tanzania. Since the existence of sub-clinically infected pigs may contribute to the persistence of the virus, our findings suggest continuous surveillance and characterization of ASFV isolates in disease-endemic regions.

Re-emergence of genotype I of African swine fever virus in Ivory Coast.

In July 2014, an outbreak of severe haemorrhagic disease in a domestic pig population, was reported in San-Pedro, the second seaport city of Ivory Coast. Animals of all age groups developed clinical signs consistent with African swine fever (ASF). Tissue and serum samples from dead pigs were sent to the laboratory for diagnostic confirmation and molecular characterization based on the partial B646L (p72), the full E183L (p54) gene and the central variable region of the B602L gene. The PCR results confirmed the outbreak of ASF. Phylogenetic analyses based on p72 and p54 sequences showed that the San-Pedro 2014 outbreak virus strain belongs to p72 genotype I. The Analysis of the tetrameric amino acid repeat regions of the B602L gene showed two repeat signatures which differ by an extra A = CAST in the second signature. The ASFV sequence of the San-Pedro 2014 outbreak strain is closely related to historical and recent ASFV strains collected in Angola and Cameroon whose ships have repeatedly visited the seaport of San-Pedro from March to June 2014. The 2014 viruses are distinct from the strains involved in the previous ASF wave in 1996 in Ivory Coast.

A novel HRM assay for the simultaneous detection and differentiation of eight poxviruses of medical and veterinary importance.

Poxviruses belonging to the Orthopoxvirus, Capripoxvirus and Parapoxvirus genera share common host species and create a challenge for diagnosis. Here, we developed a novel multiplex PCR method for the simultaneous detection and differentiation of eight poxviruses, belonging to three genera: cowpox virus (CPXV) and camelpox virus (CMLV) [genus Orthopoxvirus]; goatpox virus (GTPV), sheeppox virus (SPPV) and lumpy skin disease virus (LSDV) [genus Capripoxvirus]; orf virus (ORFV), pseudocowpox virus (PCPV) and bovine papular stomatitis virus (BPSV) [genus Parapoxvirus]. The assay is based on high-resolution melting curve analysis (HRMCA) of PCR amplicons produced using genus specific primer pairs and dsDNA binding dye. Differences in fragment size and GC content were used as discriminating power. The assay generated three well separated melting regions for each genus and provided additional intra-genus genotyping allowing the differentiation of the eight poxviruses based on amplicon melting temperature. Out of 271 poxviral DNA samples tested: seven CPXV, 25 CMLV, 42 GTPV, 20 SPPV, 120 LSDV, 33 ORFV, 20 PCPV and two BPSV were detected; two samples presented co-infection with CMLV and PCPV. The assay provides a rapid, sensitive, specific and cost-effective method for the detection of pox diseases in a broad range of animal species and humans.

Genetic Assessment of African Swine Fever Isolates Involved in Outbreaks in the Democratic Republic of Congo between 2005 and 2012 Reveals Co-Circulation of p72 Genotypes I, IX and XIV, Including 19 Variants.

African swine fever (ASF) is a devastating disease of domestic pigs. It is a socioeconomically important disease, initially described from Kenya, but subsequently reported in most Sub-Saharan countries. ASF spread to Europe, South America and the Caribbean through multiple introductions which were initially eradicated-except for Sardinia-followed by re­introduction into Europe in 2007. In this study of ASF within the Democratic Republic of the Congo, 62 domestic pig samples, collected between 2005-2012, were examined for viral DNA and sequencing at multiple loci: C-terminus of the B646L gene (p72 protein), central hypervariable region (CVR) of the B602L gene, and the E183L gene (p54 protein). Phylogenetic analyses identified three circulating genotypes: I (64.5% of samples), IX (32.3%), and XIV (3.2%). This is the first evidence of genotypes IX and XIV within this country. Examination of the CVR revealed high levels of intra-genotypic variation, with 19 identified variants.

Evaluation of the antigenic relatedness and cross-protective immunity of the neuraminidase between human influenza A (H1N1) virus and highly pathogenic avian influenza A (H5N1) virus.

To determine the genetic and antigenic relatedness as well as the cross-protective immunity of human H1N1 and avian H5N1 influenza virus neuraminidase (NA), we immunized rabbits with either a baculovirus-expressed recombinant NA from A/Beijing/262/95 (BJ/262) H1N1 or A/Hong Kong/483/97 (HK/483) H5N1 virus. Cross-reactive antibody responses were evaluated by multiple serological assays and cross-protection against H5N1 virus challenge was evaluated in mice. In a neuraminidase inhibition (NI) test, the antisera exhibited substantial inhibition of NA activity of the homologous virus, but failed to inhibit the NA activity of heterologous virus. However, these antisera exhibited low levels of cross-reactivity measured by plaque size reduction, replication inhibition, single radial hemolysis, and ELISA assays. Passive immunization with HK/483 NA-specific antisera significantly reduced virus replication and disease, and afforded almost complete protection against lethal homologous virus challenge in mice. However, passive immunization with BJ/262 (H1N1) NA-specific antisera was ineffective at providing cross-protection against lethal H5N1 virus challenge and only slightly reduced weight loss. Substantial amino acid variation among the NA antigenic sites was observed between BJ/262 and HK/483 virus, which was consistent with the lack of cross-reactive NI activity by the antibody and limited cross-protective immunity in mice. These results show a strong correlation between the lack of cross-protective immunity and low structural similarities of NA from a human seasonal H1N1 virus and an avian H5N1 influenza virus.

Neutralization of HIV subtypes A and D by breast milk IgG from women with HIV infection in Uganda.

OBJECTIVES: Among HIV-exposed infants in resource-limited countries, 8-12% are infected postnatally by breastfeeding. However, most of those uninfected at birth remain uninfected over time despite daily exposure to HIV in breast milk. Thus, we assessed the HIV-inhibitory activity of breast milk. METHODS: We measured cross-clade neutralization in activated PBMC of Ugandan subtype A (92UG031) and D (92UG005) primary HIV by breast milk or purified milk IgG and IgA from 25 HIV-infected Ugandan women. Isotype-specific antigen recognition was resolved by immunoblot. We determined HIV subtype from envelope population sequences in cells from 13 milk samples by PCR. RESULTS: Milk inhibited p24 production by ≥50% (dose-dependent) by subtype A (21/25; 84%) and subtype D (11/25; 44%). IgG consistently reacted with multiple HIV antigens, including gp120/gp41, but IgA primarily recognized p24 alone. Depletion of IgG (n = 5), not IgA, diminished neutralization (mean 78 ± 33%) that was largely restored by IgG repletion. Mothers infected with subtype A more effectively neutralized subtype A than D. CONCLUSIONS: Breast milk from HIV-infected women showed homotypic and cross-subtype neutralization of HIV by IgG-dependent and -independent mechanisms. These data direct further investigations into mechanisms of resistance against postnatal transmission of HIV to infants from their mothers.

Effect of oseltamivir carboxylate consumption on emergence of drug-resistant H5N2 avian influenza virus in Mallard ducks.

Oseltamivir carboxylate (OC) has been detected in environmental waters at various levels during recent influenza seasons in humans, reflecting levels of usage and stability of this drug. In consideration of the role of waterfowl as hosts for influenza viruses that may contribute to human infections, we evaluated the effect of consumption of low doses of OC on development of oseltamivir-resistant influenza virus mutants in mallard ducks (Anas platyrhynchos) infected with two different low-pathogenic (LP) H5N2 avian influenza viruses (AIV). We detected development of virus variants carrying a known molecular marker of oseltamivir resistance (neuraminidase E119V) in 4 out of 6 mallards infected with A/Mallard/Minnesota/182742/1998 (H5N2) and exposed to 1,000 ng/liter OC. The mutation first appeared as a minor population on days 5 to 6 and was the dominant genotype on days 6 to 8. Oseltamivir-resistant mutations were not detected in virus from ducks not exposed to the drug or in ducks infected with a second strain of virus and similarly exposed to OC. Virus isolates carrying the E119V mutation displayed in vitro replication kinetics similar to those of the wild-type virus, but in vivo, the E119V virus rapidly reverted back to wild type in the absence of OC, and only the wild-type parental strain was transmitted to contact ducks. These results indicate that consumption by wild waterfowl of OC in drinking water may promote selection of the E119V resistance mutation in some strains of H5N2 AIV that could contribute to viruses infecting human populations.

Transmission of avian influenza A viruses among species in an artificial barnyard.

Waterfowl and shorebirds harbor and shed all hemagglutinin and neuraminidase subtypes of influenza A viruses and interact in nature with a broad range of other avian and mammalian species to which they might transmit such viruses. Estimating the efficiency and importance of such cross-species transmission using epidemiological approaches is difficult. We therefore addressed this question by studying transmission of low pathogenic H5 and H7 viruses from infected ducks to other common animals in a quasi-natural laboratory environment designed to mimic a common barnyard. Mallards (Anas platyrhynchos) recently infected with H5N2 or H7N3 viruses were introduced into a room housing other mallards plus chickens, blackbirds, rats and pigeons, and transmission was assessed by monitoring virus shedding (ducks) or seroconversion (other species) over the following 4 weeks. Additional animals of each species were directly inoculated with virus to characterize the effect of a known exposure. In both barnyard experiments, virus accumulated to high titers in the shared water pool. The H5N2 virus was transmitted from infected ducks to other ducks and chickens in the room either directly or through environmental contamination, but not to rats or blackbirds. Ducks infected with the H7N2 virus transmitted directly or indirectly to all other species present. Chickens and blackbirds directly inoculated with these viruses shed significant amounts of virus and seroconverted; rats and pigeons developed antiviral antibodies, but, except for one pigeon, failed to shed virus.

H5N1 VLP vaccine induced protection in ferrets against lethal challenge with highly pathogenic H5N1 influenza viruses.

In this study, recombinant virus-like particles (VLPs) were evaluated as a candidate vaccine against emerging influenza viruses with pandemic potential. The VLPs are composed of the hemagglutinin (HA), neuraminidase (NA), and matrix 1 (M1) proteins of the H5N1 A/Indonesia/05/2005 (clade 2.1; [Indo/05]) virus, which were expressed using baculovirus in Spodoptera frugiperda (Sf9) cells. Ferrets received either 2 injections of the VLP vaccine at escalating doses (based on HA content), recombinant HA, or were mock vaccinated. Vaccinated ferrets were then challenged with either H5N1 Indo/05 or H5N1 A/Viet Nam 1203/2004 (VN/04) wild-type viruses. All ferrets that received the VLP vaccine survived regardless of the VLP dose or challenge strain, whereas seven of eight mock vaccinated ferrets died. The VLP vaccine induced HAI antibodies against the homologous H5N1 clade 2.1 strain, as well as heterologous strains from H5N1 clades 1, 2.2, and 2.3. The magnitude of the HAI titers correlated with VLP dose. Neutralizing antibody responses against the Indo/05 and VN/04 strains showed a similar pattern. Affinity of the anti-HA antibodies raised by the H5N1 Indo/05 VLPs had a higher association rate to the homologous clade 2.1 HA than to the clade 1 (VN/04) HA; however, once bound, antibodies had similar slow disassociation rates. These results provide support for continued development of the H5N1 VLPs as a candidate vaccine against pandemic influenza. Exploration of immunologic correlates of protection for H5N1 vaccines beyond HAI and neutralizing antibody responses is warranted.

CD209L (L-SIGN) is a receptor for severe acute respiratory syndrome coronavirus.

Angiotensin-converting enzyme 2 (ACE2) is a receptor for SARS-CoV, the novel coronavirus that causes severe acute respiratory syndrome [Li, W. Moore, M. J., Vasilieva, N., Sui, J., Wong, S. K., Berne, M. A., Somasundaran, M., Sullivan, J. L., Luzuriaga, K., Greenough, T. C., et al. (2003) Nature 426, 450-454]. We have identified a different human cellular glycoprotein that can serve as an alternative receptor for SARS-CoV. A human lung cDNA library in vesicular stomatitis virus G pseudotyped retrovirus was transduced into Chinese hamster ovary cells, and the cells were sorted for binding of soluble SARS-CoV spike (S) glycoproteins, S(590) and S(1180). Clones of transduced cells that bound SARS-CoV S glycoprotein were inoculated with SARS-CoV, and increases in subgenomic viral RNA from 1-16 h or more were detected by multiplex RT-PCR in four cloned cell lines. Sequencing of the human lung cDNA inserts showed that each of the cloned cell lines contained cDNA that encoded human CD209L, a C-type lectin (also called L-SIGN). When the cDNA encoding CD209L from clone 2.27 was cloned and transfected into Chinese hamster ovary cells, the cells expressed human CD209L glycoprotein and became susceptible to infection with SARS-CoV. Immunohistochemistry showed that CD209L is expressed in human lung in type II alveolar cells and endothelial cells, both potential targets for SARS-CoV. Several other enveloped viruses including Ebola and Sindbis also use CD209L as a portal of entry, and HIV and hepatitis C virus can bind to CD209L on cell membranes but do not use it to mediate virus entry. Our data suggest that the large S glycoprotein of SARS-CoV may use both ACE2 and CD209L in virus infection and pathogenesis.

Detection and quantitation of bovine respiratory syncytial virus using real-time quantitative RT-PCR and quantitative competitive RT-PCR assays.

A single tube, fluorogenic probe-based, real-time quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR) assay was developed for detection and quantitation of bovine respiratory syncytial virus (BRSV) using BioRad's iCycler iQ. Real-time Q-RT-PCR was compared with quantitative competitive RT-PCR (QC-RT-PCR) and viral titers. Viral mRNA levels were measured in BRSV-infected bovine turbinate cell lysate harvested at eight time points (1.5, 6, 12, 24, 36, 48, 60, 72 h) post-infection. A homologous BRSV cRNA standard was used for quantitation of the mRNA by plotting a standard curve of cycle threshold (Ct) values versus standard 10-fold dilutions of cRNA of known concentrations. Detection as low as 171 copies/microl of standard BRSV cRNA was possible. For QC-RT-PCR, a competitor RNA molecule having a deletion was designed and used for quantitation of the BRSV viral mRNA. The results of real-time Q-RT-PCR and QC-RT-PCR assays showed a positive correlation. Real-time Q-RT-PCR was a sensitive, specific, rapid, and efficient method that eliminates the post-PCR processing steps when compared to QC-RT-PCR. Quantitation of BRSV using real-time Q-RT-PCR will have application in studies aimed at understanding the pathogenesis of BRSV.