The oncolytic parvovirus H-1PV : tumor-suppressing mechanisms

H-1 parvovirus (H-1PV) är ett autonomt onkolytiskt parvovirus. Dess naturliga värddjur är råtta, men det kan även infektera andra vertebrater. H-1PV kan tas upp av alla celler men är endast skadligt för prolifererande celler i neonatala djur och tumöromvandlade celler. Vid infektion av tumörceller kan tumörsupprimerande mekanismer genereras. Dessa kan vara direkt cytotoxiska och celldödande, eller indirekta genom att aktivera viralt eller tumörspecifikt immunsvar. Under de senaste två decennierna har många framgångsrika prekliniska studier genomförts men de tumörsupprimerande mekanismerna för H-1PV är ännu långt ifrån klarlagda. Den här litteraturstudien syftar till att beskriva några av de egenskaper och mekanismer som är av betydelse för H-1PVs tumörsuppression. Grundförutsättningen för H-1PVs tumörsupprimerande effekt är dess onkotropism, det vill säga att virusets livscykel gynnas av den cellulära miljön som råder i tumörceller. Vid viral replikation och produktion av virala proteiner induceras cytotoxiska mekanismer. Dessa mekanismer varierar mellan olika celltyper. Exempel på cytotoxiska mekanismer som observerats i H-1PV-infekterade tumörceller är cellcykelarrest, aktivering av kaspaser, ackumulering av reaktiva syreföreningar och ackumulering av lysosomala enzymer. Exempel på H-1PV-inducerad immunförsvarsaktivering är aktivering av dendritiska celler och NK-celler (natural killer cells). Hitintills har H-1PV-forskningen fokuserat på behandlingsmöjligheter för humana tumörer, men då andra däggdjur i likhet med människa saknar tidigare antiviral immunitet för H-1PV och har stora fysiologiska likheter med människan är det inte orimligt att anta att H-1PV kan vara en kandidat för tumörbehandling även inom veterinärmedicinen. Exempel på humana tumörtyper som svarat väl på H-1PV-infektion är glioblastoma multiforme, hepatom, lymfom, melanom, och carcinom i pankreas, bröst, mage, colon och cervix. År 2011 inleddes den första kliniska studien för H-1PV. Den studien pågår ännu och syftar till att studera säkerheten och effektiviteten av H-1PV som behandling mot progressiv glioblastoma multiforme. Den främsta fördelen med H-1PV gentemot befintliga behandlingsalternativ är att H-1PV inte ger några biverkningar. Det finns anledning att tro på H-1PV som ett framtida behandlingsalternativ mot tumörer, men än finns mycket kvar att lära om viruset.Parvovirus H-1 (H-1PV) is an autonomous oncolytic parvovirus. Its natural host is rat, but it can also infect other vertebrates. Cellular virus uptake is possible for all cells but virus infection is only harmful for transformed cells and proliferating cells in neonatal animals. Infection of transformed cells can induce tumor suppressing mechanisms. These mechanisms consist of intracellular cytotoxic mechanisms, induced cell death, and activation of antiviral and tumor specific immunity. During the last two decades pre-clinical studies with promising results have been performed, however there is still much to be clarified about the tumor suppressing properties of H-1PV. This bachelor thesis aims to describe some of the tumor suppressing mechanisms that so far have been indicated for H-1PV. The first reason for the tumor suppression of H-1PV is that H-1PV is oncotropic, which means that the intracellular environment in transformed cells is in favor for the viral life cycle. Viral replication and production of viral proteins induces cytotoxic mechanisms. These mechanisms vary among different cell types. Examples of cytotoxic mechanism that have been observed in H-1PV-infected tumor cells are cell cycle arrest, activation of caspases, accumulation of reactive oxygen species and accumulation of lysosomal enzymes. Examples of H-1PV-induced activation of the immune system are activation of dendritic cells and natural killer cells. So far the research of H-1PV has been focusing on treatment of human tumors, and since other mammals have much the same physiology as humans and in similarity to humans lack preexisting antiviral immunity for H-1PV, it is likely that H-1PV can become a candidate as a antitumor therapy also within the field of veterinarian medicine. Examples of human tumors in which H-1PV has proved efficiency in pre-clinical studies are glioblastoma multiforme, hepatoma, lymphoma, melanoma, and pancreatic, mammary, gastric, colon and cervical carcinoma. In 2011 the first clinical trial for H-1PV was launched. That study is still ongoing and aims to find out about the safety and efficacy of H-1PV-treatment for patients with recurrent glioblastoma multiforme. The uppermost advantage of H-1PV compared to other existing therapies is that H-1PV is non-pathogenic. With further studies it is not unlikely that H-1PV will take a role as an alternative antitumor therapy

Longitudinal study of the immune response and memory following natural bovine respiratory syncytial virus infections in cattle of different age

Human and bovine respiratory syncytial virus (HRSV and BRSV) are closely genetically related and cause respiratory disease in their respective host. Whereas HRSV vaccines are still under development, a multitude of BRSV vaccines are used to reduce clinical signs. To enable the design of vaccination protocols to entirely stop virus circulation, we aimed to investigate the duration, character and efficacy of the immune responses induced by natural infections. The systemic humoral immunity was monitored every two months during two years in 33 dairy cattle in different age cohorts following a natural BRSV outbreak, and again in selected individuals before and after a second outbreak, four years later. Local humoral and systemic cellular responses were also monitored, although less extensively. Based on clinical observations and economic losses linked to decreased milk production, the outbreaks were classified as moderate. Following the first outbreak, most but not all animals developed neutralising antibody responses, BRSV-specific IgG1, IgG2 and HRSV F- and HRSV N-reactive responses that lasted at least two years, and in some cases at least four years. In contrast, no systemic T cell responses were detected and only weak IgA responses were detected in some animals. Seronegative sentinels remained negative, inferring that no new infections occurred between the outbreaks. During the second outbreak, reinfections with clinical signs and virus shedding occurred, but the signs were milder, and the virus shedding was significantly lower than in naïve animals. Whereas the primary infection induced similar antibody titres against the prefusion and the post fusion form of the BRSV F protein, memory responses were significantly stronger against prefusion F. In conclusion, even if natural infections induce a long-lasting immunity, it would probably be necessary to boost memory responses between outbreaks, to stop the circulation of the virus and limit the potential role of previously infected adult cattle in the chain of BRSV transmission

Evaluation of the economic impact and description of a BRSV-outbreak in a dairy herd

Bovine respiratory syncytial virus (BRSV) is globally one of the most important causes of respiratory disease in beef and dairy cattle of all ages and results in considerable costs for cattle farmers and significant suffering for the affected animals. The BRSV-infection can be subclinical to severe, and even fatal. There is no effective treatment and no commercially available vaccine that induces satisfying duration of protection, though promising vaccine candidates are under development. In addition to treatment costs and animal loss BRSV is associated to reduced production, such as reduced growth, reduced milk yield and fertility disorders. The virus can be transmitted directly and indirectly by respiratory secretion. Spread between and within herds is often rapid. The contagious nature of the virus together with its severe impact on animal welfare and economy necessitate effective preventive measures. To develop cost-efficient prevention strategies against BRSV evaluation of the economic impact caused by the virus is necessary. This study aimed to estimate the economic impact of BRSV by analyzing the short-term costs associated to a BRSV-outbreak in a Swedish dairy herd. The parameters investigated were veterinary care, drug treatment, diagnostic tests, milk production, death and extra labor. The sum of the estimated budget items was 71,464 SEK, which rounded corresponded to 130 SEK per animal housed in the farm during the outbreak, or 270 SEK per cow. Out of the investigated costs milk loss (23,234 SEK, 32,5%), death (21,000 SEK, 29,4%) and extra personnel (17,112 SEK, 23,9%) were the three major budget items (61,346 SEK, 85,8%). Veterinary cost (2656 SEK, 3,7%), medicine costs (5399 SEK, 7,6%) and costs for diagnostic tests (2063 SEK, 2,9 %) were comparably small costs (10,118 SEK, 14,2%). Due to the possible long-term consequences such as reduced growth and reproduction disorders not being included, the estimated sum of 71,464 SEK is most likely an underestimation of the true cost for the farm. The estimated cost in this study is an example of the short-term economic impact of a BRSVoutbreak. By seemingly small changes of an outbreak’s characteristics the economic impact can change greatly. The investigated outbreak in the present study was for example characterized by relatively low mortality, 0,37%. In literature outbreaks of up to 20 % mortality are described. The clinical illness in the present herd was also relatively low, which was explained mainly by high inter-herd biosecurity and overall good cattle management and air quality resulting in minimized viral exposure and consequently less severely affected animals. The economic estimations and the epidemiologic observations presented in this study are in agreement with other papers emphasizing the economic impact and animal suffering of BRSV. Further, the epidemiologic observations presented in this study indicates that decreased feed consumption and decreased milk yield can be used as early warning signs of an upcoming BRSV-outbreak. In conclusion more research is needed to gain broader and more in-depth knowledge about the economic impact of BRSV, both for different kinds of herds and in terms of long-term economic consequences.Bovint respiratoriskt syncytialt virus (BRSV) är globalt en av de viktigaste orsakerna till luftvägssjukdom hos nötkreatur av alla åldrar och resulterar i stora kostnader för nötbönder och avsevärt lidande för de drabbade djuren. En BRSV-infektion kan vara subklinisk till allvarlig, och har i vissa fall dödlig utgång. Det finns ingen effektiv behandling och kommersiellt tillgängliga vaccin skyddar inte optimalt. Lovande vaccinkandidater är dock under utveckling. Utöver vårdkostnader och djurförluster leder BRSV till försämrad produktion, så som i form av minskad tillväxt, sänkt mjölkproduktion och reproduktionsstörningar. Viruset kan smitta direkt eller indirekt via sekret från luftvägarna. Spridning mellan och inom besättningar sker ofta snabbt. På grund av virusets smittsamma natur i kombination med dess negativa påverkan på djurvälfärd och bondens ekonomi är behovet av effektiva förebyggande åtgärder stort. För att möjliggöra framtagandet av kostnadseffektiva preventionsstrategier är det nödvändigt att kvantifiera kostnaderna associerade till BRSV. Den här studien syftar till att bidra med information om BRSVs påverkan på lantbrukets ekonomi genom att analysera de kortsiktiga ekonomiska effekterna av ett BRSV-utbrott i en svensk mjölkkobesättning. Parametrarna som undersöktes i den här studien var veterinärvård, läkemedelskostnader, diagnostiska tester, mjölkproduktion, dödsfall och extra arbete. Totalsumman för dessa utgifter uppgick till 71 464 kronor, vilket avrundat motsvarade 130 kronor per djur i besättningen. De tre största utgiftsposterna var mjölkförlust (23 234 kronor, 32,5 %), dödsfall (21 000 kronor, 29,4 %) och extra personal (17 112 kronor, 23,9 %). Veterinärvård (2656 kronor, 3,7 %), läkemedelskostnader (5399 kronor, 7,6 %) och kostnader för diagnostik (2063 kronor, 2,9 %) var förhållandevis små utgifter (10 118 kronor, 14,2 %). På grund av att potentiella långtidseffekter så som minskad tillväxt och reproduktionsstörningar inte inkluderades är det troligt att kostnadsberäkningen i studien är en underskattning av gårdens verkliga totalkostnad av utbrottet. Den framtagna totalkostnadsuppgiften i den här studien är ett exempel på vad ett BRSV-utbrott kan kosta. Med till synes små förändringar i ett utbrotts karaktär kan de korrelerande ekonomiska följderna förändras stort. Utbrottet i den här studien karaktäriserades exempelvis av en förhållandevis låg mortalitet, 0,37 %. I litteraturen finns utbrott med upp till 20 % mortalitet beskrivna. Vidare var den kliniska sjukdomsgraden i det undersökta utbrottet förhållandevis låg, vilket framförallt förklarades med gårdens strikta hygienrutiner och överlag goda djurhållning och luftkvalitet, som antogs resultera i en under omständigheterna låg virusexponering och motståndskraftiga djur, och därigenom mindre allvarligt påverkade djur. De ekonomiska estimeringarna och de epidemiologiska observationerna i den här studien stödjer de i litteraturen tidigare angivna observationerna för BRSVs negativa konsekvenser gällande ekonomi och djurvälfärd. Vidare indikerar de epidemiologiska observationerna i den här studien att nedsatt aptit och sänkt mjölkproduktion kan användas som tidiga varningstecken för ett nära förestående BRSV-utbrott. Fortsatt forskning är nödvändig för att bredda och fördjupa kunskapen om BRSVs ekonomiska konsekvenser, både för olika besättningstyper och gällande långtidskonsekvenser

Longitudinal study of the immune response and memory following natural bovine respiratory syncytial virus infections in cattle of different age

International audienceHuman and bovine respiratory syncytial virus (HRSV and BRSV) are closely genetically related and cause respiratory disease in their respective host. Whereas HRSV vaccines are still under development, a multitude of BRSV vaccines are used to reduce clinical signs. To enable the design of vaccination protocols to entirely stop virus circulation, we aimed to investigate the duration, character and efficacy of the immune responses induced by natural infections. The systemic humoral immunity was monitored every two months during two years in 33 dairy cattle in different age cohorts following a natural BRSV outbreak, and again in selected individuals before and after a second outbreak, four years later. Local humoral and systemic cellular responses were also monitored, although less extensively. Based on clinical observations and economic losses linked to decreased milk production, the outbreaks were classified as moderate. Following the first outbreak, most but not all animals developed neutralising antibody responses, BRSV-specific IgG1, IgG2 and HRSV F- and HRSV N-reactive responses that lasted at least two years, and in some cases at least four years. In contrast, no systemic T cell responses were detected and only weak IgA responses were detected in some animals. Seronegative sentinels remained negative, inferring that no new infections occurred between the outbreaks. During the second outbreak, reinfections with clinical signs and virus shedding occurred, but the signs were milder, and the virus shedding was significantly lower than in naïve animals. Whereas the primary infection induced similar antibody titres against the prefusion and the post fusion form of the BRSV F protein, memory responses were significantly stronger against prefusion F. In conclusion, even if natural infections induce a long-lasting immunity, it would probably be necessary to boost memory responses between outbreaks, to stop the circulation of the virus and limit the potential role of previously infected adult cattle in the chain of BRSV transmission

Kinetics of BRSV-neutralising serum antibody titres in cattle of different age and production status.

Cattle were (A) 23–30 months old (6.5 months gestation), (C) 7–11 months old, (D) 4–5 months old, (E) 2–3 months old, not analysed (N.A), or (F) born during or just after a BRSV outbreak in January 2016 (month 0). Limit of detection in the virus neturalising assay: titre 20.</p

Kinetics of HRSV-F-reactive serum antibodies in cattle of different age and production status.

At the time of a BRSV outbreak the cattle were (A) 23–30 months old (6.5 months gestation), (C) 7–11 months old, (D) 4–5 months old, (E) 2–3 months old, or (F) born during or just after the outbreak in January 2016 (month 0). Data are presented as competition percentage. The limit of detection is presented as a dotted line.</p

Kinetics of BRSV-specific total IgG1 in milk from cattle of different age and production status.

At the time of a BRSV outbreak (in January 2016, month 0) the cattle were (A) 23–30 months old (6.5 months gestation), or (C) 7–11 months old. Corrected optical density (COD) values are presented as percentage of a positive control serum. Broken lines represent dry periods and red circles are timepoints for calving.</p

Least Square Means of milk production, roughage and non-roughage intake of 272, 163 and 272 cows, respectively, that remained in production for commercialisation, before, during and after a BRSV outbreak in 2016.

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Kinetics of BRSV-specific serum IgG1 in cattle of different age and production status.

At the time of a BRSV outbreak the cattle were (A) 23–30 months old (6.5 months gestation), (C) 7–11 months old, (D) 4–5 months old, (E) 2–3 months old, or (F) born during or just after the outbreak in January 2016 (month 0). Corrected optical density (COD) values are presented as percentage of a positive control serum.</p

Kinetics of HRSV N-specific serum IgG1 antibodies in cattle of different age and production status.

At the time of a BRSV outbreak the cattle were (A) 23–30 months old (6.5 months gestation), (C) 7–11 months old, (D) 4–5 months old, (E) 2–3 months old, or (F) born during or just after the outbreak in January 2016 (month 0). For each serum sample, the optical density (OD) against a control antigen was subtracted from the OD value against the N protein (Corrected OD, COD) and the COD was transformed into a sample-to-positive value (SP) by using the formula SP = CODsample/(CODpos—CODneg).</p